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Pathogenic micro-organisms.A text-book 


Cornell University 


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PATHOGENIC MICRO-ORGANISMS 


MACNEAL 


PATHOGENIC 
MICRO-ORGANISMS 


A TEXT-BOOK OF 
MICROBIOLOGY FOR PHYSICIANS 
AND STUDENTS OF MEDICINE 


BY 


WARD J. ONE eee D. le me 


PROFESSOR OF PATHOLOGY AND BACT LABORATORIES 
IN THE NEW YORK POST-GRADUATE MEDICAL SCHOOL AND See NEW YORK 


(Based Upon Williams’ Bacteriology) 


-WITH 221 ILLUSTRATIONS 


SECOND EDITION, REVISED AND ENLARGED 


PHILADELPHIA 
P. BLAKISTON’S SON & CO. 
1012 WALNUT STREET 


CopyricHT, 1920, BY P. Biaxiston’s Son & Co. 


THE MAPLE PRESS YORK PA 


PREFACE TO THE SECOND EDITION 


In the preparation of this second edition the text has been re- 
vised throughout, but the changes have been in the nature of 
minor corrections and the addition of new matter to bring the text 
up to date. The general plan of the book has been preserved, 
keeping in mind its purpose as an introduction to the study of 
pathogenic micro-organisms. 

Subjects in controversy, such as the pathogenic réle of Bacillus 
influenze or of Bacillus typhi-exanthematici, have received only 
brief mention, but some references to the literature have been 
given for the possible use of more advanced students. On the 
other hand more conclusive advances in microbiology, such as the 
recent studies on botulism, yellow fever, rat-bite fever and trench 
fever have been included in the text. Several new illustrations 
have been added, the credit for which is indicated in each instance. 

The index and the table of contents have been made quite full 
in order to render the text readily accessible and to present to the 
student a skeleton outline for the review of each chapter. 

My thanks are again due to Mrs. MacNeal for assistance, 
especially in the preparation of the index. 

W. J. MacNEat 


New York 


TABLE OF CONTENTS 


INTRODUCTION 


Bacteriology and Microbiology, 1; Biological relationships, 3 ; Spontaneous genera- 
tion, 3; Heterogenesis, 4; Systematic relationships, 5; Fermentation and Putre- 
faction, 5; Specific fermentations, 6; Pathology and Hygiene, 7; Contagion, 8; 
Specific infection, 10; Antisepsis, 11; Proof of the germ theory, 11; Immunity, 
12; Parasitic protozoa, 12; Insect transmission, 13; Pathogenic spirochetes, 13; 
Filterable viruses, 13; Agriculture, 14; Biological view-point in the study of 
micro-organisms, 14. ~ , 


PART I. BACTERIOLOGICAL TECHNIC 


CHAPTER I.—THE MIcRoscoPpE AND Microscopic METHODS 


Development of the microscope, 15; Lenses, 15; Achromatic and apochromatic 
objectives, 15; Ultra-microscope and dark-field microscopy, 16; Tandem micro- 
scope, 16; Principle of the microscope, 16; Pin-point aperture, 16; Relations of 
magnification, definition and brilliancy of image, 16; Lens-armed aperture, 17; 
Two lenses in series, 18; Magnification measured by the: ratio of the opening 
and closing angles of a beam, 19; Simple microscope, 19; Reading glass, 19; 
Spherical aberration, 20; Chromatic aberration, 20; Diffraction, 20; Image 
formation in compound microscope, 22; Numerical aperture, 23; Illumination 
by the Abbé condenser, 24; Central illumination, 24; Dark-field, 25; Illumina- 
tion by broad converging beam, 25; Visibility of microscopic objects, 25; Defini- 
tion by light and shade, 26; The color picture, 28; The Bacteriological micro- 
scope, 29; Eye-pieces and objectives, 30; Use of the microscope, 31; Microscopic 
measurements, 31; The platinum wire, 32; Pasteur pipettes, 33 ; The hanging- 
drop, 34; Motility of micro-organisms, 35; Brownian motion, 35; Hanging- 
block, 35; Slide for dark-field study, 36; Use of dark-field, 36; Smear prepara- 
tions, 37; Cover-glasses, 37; Technic, 37; Slide smears, 39; Staining solutions, 
40; Aniline stains, 40; Method of simple staining, 44; Gram’s stain, 45; Acid- 
proof staining, 47; Sputum staining, 47; Spore staining, 51; Capsule stain, 52 ; 
Staining of flagella, 52 ; Wet fixation, 54; Iron hematoxylin, 54; Blood films, 55; 
Staining of tissue sections, 55; Celloidin, 55; Paraffin, 56; Sectioning, 57; 
Simple staining, 58; Gram-Weigert method, 59; Tubercle bacilli, 60; Nuclear 
stains, 61. 

vii 


t 


vill TABLE OF CONTENTS 


CHAPTER II. SrTeERILizATION, DISINFECTION, ANTISEPSIS, Foob 
PRESERVATION 


Definitions, 62; Physical sterilization, 62; Mechanical removal, 62; Desiccation, 63; 
Light, 63; Cold, 64; Heat, 64; Electricity, 71; Chemical sterilization, 71; Soaps 
71; Acids, 71; Alkalies, 73; Oxidizing agents, 73; Inorganic salts, 74; Organic 
poisons, 76; Antiseptics and preservatives, 78; Physical, 79; Chemical, 79; 
Testing of antiseptics and disinfectants, 80. 


Cuapter III. Cutrure MeEpia 


Definition, 83; Glass-ware, 83; The common media, 84; Nutrient broth, 84; Titra- 
tion of media, 85; Gelatin, 90; Agar, 92; Modifications, 92; Sterilizable special 
media, 93 ; Potato, 93; Milk, 94; Peptone solution, 94; Nitrate broth, 95; Blood- 
serum, 95; Loeffler’s blood-serum, 96; Eggs, 96; Dorset’s egg, 97; Bread paste, 
97; Media containing uncooked protein, 97; Sterile blood, 97; Ascitic fluid, 99; 
Sterilization, 100; Sterile tissue, 100; Blood-streaked agar, 101; Blood-agar, 
1o1; Broth containing tissues, 101; Ascitic-fluid agar, 101; Ascitic fluid with 
tissue, 101; Other special media, 102. 


CHAPTER IV. COLLECTION OF MATERIAL FOR BACTERIOLOGICAL 
; STUDY 


General considerations, 103; Sampling water and foods, 103; Material from the 
body, 103 ; Sputum, 104; Urine, 104; Blood and transudates, 104; Cerebro-spinal 
fluid, 105; Feces and intestinal juice, 105; Pus and exudates, 106; Material from 
autopsies, 107. 


CHAPTER V. THE CULTIVATION OF MICRO-ORGANISMS 


Avoidance of contamination, 108; Isolation of bacteria, 109; Plate cultures, 110; 
Roll tubes, 114; Streak method, 116; Tall-tube method, 116; Colonies, 117; 
Pure cultures, 117; Stock cultures, 118; Regulation of temperature, 119; High 
temperature incubator, 119; Gas-regulator, 120; Automatic safety-burner, 123; 
Incubator room, 124; Prevention of drying, 124; Low-temperature incubator, 
125; Cultivation of anaerobic bacteria, 128; Deep stab, 128; Veillon tall-tube 
method, 129; Fermentation tube, 129; Removal of oxygen, 129; Hydrogen at- 
mosphere, 130; Further methods, 134. : 


CuaprerR VI. Meruops or ANIMAL EXPERIMENTATION 


Value of animal experimentation, 135; Care of animals, 135 ; Holding for operation, 
136; Inoculation, 137; Subcutaneous and intraperitoneal, 137; Intracranial, 
137; Into circulating blood, 137; Other sites, 138; Subcutaneous application, 
138; Alimentary and respiratory infection, 138; Collodion capsules,-138 ; Obser- 
vation of infected animals, 140; Post-mortem examination, 140. 


TABLE OF CONTENTS ix 


PART II. GENERAL BIOLOGY OF MICRO-ORGANISMS 


CHAPTER VII. MorpHoLocy AND CLASSIFICATION 


Molds and yeasts, 143; Bacteria, 147; Trichobacteria, 147; Spherical bacteria, 148; 
Cylindrical bacteria, 150; Spiral bacterial, 152; Structure of the lower bacteria, 

- 153; Endospores, 156; Filterable viruses, 156; Protozoa, 156; Flagellates, 157; 
Rhizopods, 161; Sporozoa, 161; Ciliates, 166; Outline classification of micro- 
organisms, 166; Specific nomenclature, 167. 


Cuapter VIII. Puysiotocy or MICRO-ORGANISMS 


Relations of morphology and physiology, 169 ; Conditions of physiological study, 170; 
Environmental factors, 171; Moisture, 171; Organic food, 171; Inorganic salts 
and chemical reaction, 172; Oxygen, 173; Temperature, 173; Microbic variation, 
174; Products of microbic growth, 175; Physical effects, 175; Chemical effects, 
175; Enzymes, 176; Toxins, 178; Relation of microbe and its environment, 178; 
Morphological characters, 178; Physiological tests, 180; Descriptive chart of 
the Society of American Bacteriologists, 180. 


CHAPTER IX. Tue DISTRIBUTION OF MICRO-ORGANISMS AND THEIR 
RELATION TO SPECIAL HABITATS 


General distribution, 181; Micro-organisms of the Soil, 182; Pathogenic soil 
bacteria, 183; Micro-organisms of the air, 183 ; Micro-organisms of Water and 
Ice, 185; Self-purification of water, 186; Storage of water, 187; Filtration, 187; 
Disinfection of water, 189; Bacteriological examination of water, 189; Detec- 
tion of intestinal bacteria, 193; Bacteriological examination of ice, 195; Micro- 
organisms of food, 196; Milk, 196; Milk flora, 197; Pathogenic microbes in 
milk, 199; Milk for infant feeding, 199; Other foods, 200. 


CHAPTER X. PARASITISM AND PATHOGENESIS 


The parasitic relation, 201; Pathogenesis, 202; Rules of Koch, 202; Infectious 
disease, 203; Possibility of infection, 203; Susceptibility and resistance, 203; 
Number of invaders, 204; Modes of introduction, 204; Local susceptibility, 206: 
Local and general infections, 206; Transmission of infection, 207; Healthy 
carriers of infection, 208. 


CHAPTER XJ. THE PATHOGENIC PROPERTY OF MICRO-ORGANISMS 
Adaptation to parasitism, 209; Virulence, 209; Microbic poisons, 210; Defensive 
mechanisms, 211. 
CHAPTER XII.. REACTION OF THE Host To INFECTION 


Facts and theories, 213 ; Physiological hyperplasia, 213 ; Phagocytosis and encapsu- 
lation, 214; Chemical constitution of the cell, 214; Antitoxins, 215; Cell re- 
ceptor of first order, 216; Precipitins, 216; Receptor of second order, 217; 


x TABLE OF CONTENTS 


Agglutinins, 218; Phenomenon of agglutination, 218; Bactericidal substances, 
219; Cytolysins, 220; Receptor of third order, 221; Amboceptor and comple- 
ment, 221; Deviation of complement, 222; Fixation of complement, 223; 
Opsonins, 224; Anti-aggressins, 225; Source and distribution of antibodies, 
225; Allergy, 226. 


CHAPTER XIII. Immunity AND HyYPERSUSCEPTIBILITY. THEORIES 
or IMMUNITY 


Immunity, 227; Natural immunity, 227; Immunity of species, 227. Racial im- 
munity, 228; Individual variations, 228; Acquired immunity, 229; Active 
immunity, 229; Passive immunity, 231. Combined active and passive 
immunity, 232; Mechanisms of immunity, 232; Hypersusceptibility or Ana- 
phylaxis, 233; Mechanisms of immunity, 234. 


PART III. SPECIFIC MICRO-ORGANISMS 


CHaPteR XIV. Tue MoLps AND YEASTS AND DISEASES CAUSED BY 
THEM 


Mucors, 237; Aspergilli, 239; Penicillium crustaceum, 240; Claviceps purpurea, 
240; Ergotism, 241; Saccharomyces cerevisia, 241; Coccidioides immitis, 
242; Botrytis bassiana, 242; Muscardine, 242; Oidium lactis, 244; Oidium 
albicans, 245; Thrush, 245; Monilia psilosis, 247; Achorion schoenleinii, 
247; Favus, 247; Microsporon audouini, 250; Alopecia areata, 250; Micro- 
sporon furfur, 250; Tricophyton acuminatum, 250; Sporotrichum schencki, 
251; Sporotrichum beurmanni, 252; Cryptococcus gilchristi, 252; Blasto- 
mycosis, 253. 

CHAPTER XV. TRICHOMYCETES 


Actinomyces bovis, 254; Streptothrix madurz, 256; Mycetoma, 256; Streptothrix 
putorii, 257; Cladothrix, 257; Leptothrix buccalis, 257. 


CuapTteR XVI. THE CoccacE& anpD THEIR PaRASsITIC 
RELATIONSHIPS 


Diplococcus gonorrhee, 258; Occurrence, 258; Culture, 258; Toxins, 260; Gonor- 
rhea, 260; Specific diagnosis, 261; Prophylaxis, 261; Diplococcus meningi- 
tidis, 262; Anti-meningococcus serum, 263; Quincke’s puncture, 264; Exami- 
nation of spinal fluid, 264; Diagnosis, 265; Diplococcus catarrhalis, 265; Dip- 
lococcus penumonia, 266; Occurrence, 266; Morphology, 266; Cultures, 
267; Pneumonia, 268; Toxins, 268; Immunity, 268; Type determination, 269; 
Streptococcus viridans, 270; Streptococcus pyogenes, 271; Occurrence, 271; 
Cultures, 272; Animal inoculation, 273; Surgical infections, 273; Erysipelas, 273; 
Puerperal fever, 274; Immunity, 274; Streptococcus lacticus, 275; Staphy- 
lococcus aureus, 275; Occurrence, 275. Morphology, 275; Cultures, 275; 
Toxins, 276; Animal inoculation, 277; Infection of man, 277; Immunity, 277; 
Vaccine therapy, 277; Staphylococcus albus, 277; Micrococcus tetragenus, 
278; Sarcina ventriculi, 278; Sarcina aurantiaca, 278; Micrococcus agilis, 278. 


TABLE OF CONTENTS xi 


CHAPTER XVII. Bacirrace#: Tue Sporccrenrc AErROBES 


Bacillus mycoides, 279; Bacillus vulgatus, 279; Bacillus subtilis, 280; Parasitism 
280; Bacillus anthracis, 281; Occurrence, 281; Morphology, 282; Resistance 
283; Anthrax, 283; Human anthrax, 284; Immunity, 284; Serum, 285. 


CHaPtER XVIII. Bacttracem: Tue Sporocentc ANAEROBES 


Group characters and habitat, 286; Clostridium edematis, 275; Putrefactive prop- 
erties, 287; Malignant edema, 287; Clostridium feseri, 287; Clostridium per- 
fringens, 287; Occurrence, 287; Characters, 288; Emphysematous gangrene, 
289; Clostridium tetani, 290 ; Occurrence, 290; Morphology, 290; Cultures, 290; 
Toxin, 291; Tetanus, 291; Immunity, 292; Antitoxin, 293; Standard unit, 293, 
Prophylaxis and treatment, 295; Clostridium botulinum, 295; Botulin, 296; 
Immune serum, 296; Botulism, 296. 


CHAPTER XIX. MycoBAcTERIACEZ: THE BACILLUS OF DIPHTHERIA 
AND OTHER SPECIFIC BACILLI PARASITIC ON SUPERFICIAL Mucous 
MEMBRANES 


Bacillus diphtheriz, 298; Occurrence, 298; Culture, 298; Toxin, 301; Diphtheria, 
302; Bacteriological diagnosis, 303; Transmission of the disease, 305; Immu- 
nity, 305; Antitoxin, 306; Standard unit of antitoxin, 307; Prophylactic and 
therapeutic use of antitoxic serum, 308; Untoward effects, 308; Schick reaction, 
309; Bacillus xerosis, 309; Bacillus hoffmanni, 309; Morax-Axenfeld bacillus, 
309; Koch-Weeks bacillus, 309; Bacillus pertussis, 309; Bacillus influenze, 
311; Bacillus chancri, 312. 


~ 


CHAPTER XX. MyYCOBACTERIACEZ: THE TUBERCLE BACILLUS AND 
OTHER ACID-PROOF BACTEPIA 


Bacillus tuberculosis, 313; Human type, 314; Occurrence, 314; Morphology, 314; 
Cultures, 315; Chemical composition, 316; Toxins, 317; Resistance, 318; Tuber- 
culin, 318; Animal inoculation, 319; Tuberculosis, 319; The tubercle, 320; 
Mode of transmission, 321; Bacteriological diagnosis, 321; Allergic reactions, 
322; Bovine type, 324; Avian type, 325; Fish or amphibian type, 326; Bac- 
illus lepre, 326; Morphology and occurrence, 326; Leprosy, 326; Bacillus 
smegmatis, 327; Bacillus moelleri, 327; Other acid-proof organisms, 328; 
Pseudo-bacilli, 328. : 


CHAPTER XXI. BACTERIACEZ: THE BACTERIA OF THE HEMoR- 
RHAGIC SEPTICAMIAS, OF PLAGUE AND OF MALTA FEVER 


Bacillus avisepticus, 329; Bacillus plurisepticus, 330; Bacillus pestis, 330; Occur- 
rence and morphology, 331; Cultures, 331; Toxins, 331; Animal inoculation, 
332; Bubonic plague, 332; Epizoétic plague, 333; Human plague, 333; Immu- 


xii TABLE OF CONTENTS 


nity, 333; Immune serum, 334; Prophylaxis, 334; Eradication of endemic 
centers, 334; Bacillus melitensis, 334; Malta fever, 335. 


CHAPTER XXII. BacTertackE#: THE CoLon, TYPHOID AND 
DvYSENTERY BACILLI 


Bacillus coli, 337; Occurrence and morphology, 337; Cultures, 338; Pathogenic 
relations, 339; Bacillus aerogenes, 339; Bacillus pneumonie, 340; Bacillus 
rhinoscleromatis, 340; Bacillus entertidis, 341; Bacillus suipestifer, 342; 
Bacillus psittacosis, 342; Bacillus typhi murium, 342; Bacillus alkaligenes, 
342; Bacillus paratyphosus, 342; Bacillus typhosus, 343 ; Occurrence and mor- 
phology, 343; Cultures, 344; Resistance, 345; Toxins, 345; Animal inoculation, 
345; Typhoid fever, 346; Bacterial diagnosis, 346;. Transmission of the disease, 
349; Prevention, 350; Bacillus dysenteriz, 351; Epidemic dysentery, 351; 
Paradysentery bacilli, 352. 


CHAPTER XXIII. BAcTERIACER: BactitLus MALLEI AND MISCEL- 
LANEOUS BACILLI 


Bacillus mallei, 354; Occurrence and morphology, 354; Cultures, 354; Mallein, 
355; Glanders, 355; Bacteriological diagnosis, 355; Bacillus abortus, 356; 
Bacillus acne, 357; Bacillus fusiformis, 357 ; Bacillus bifidus, 358 ; Bacillus bul- 
garicus, 358; Bacillus vulgaris, 358; Bacillus pyocyaneus, 359; Bacillus fluo- 
rescens, 359; Bacillus violaceus, 359; Bacillus cyanogenus, 359; Bacillus 
prodigiosus, 359. 


CHAPTER XXIV. SPIRILLACEZ AND THE DISEASES CAUSED BY THEM 


Spirillum rubrum, 360; Spirillum cholerz, 360; Occurrence and morphology, 360; 
Cultures, 360; Animal inoculation, 362; Toxins, 363; Pfeiffer’s phenomencn, 
363; Asiatic cholera, 363; Mode of infection, 364; Bacteriological diagnosis, 
365; Prophylaxis, 366; Spirillum metchnikovi, 367; Spirillum Finkler-Prior, 
367; Spirillum tyrogenum, 367. 


CHAPTER XXV. SPIROCHATE 


Spirocheta plicatilis, 368; Other saprophytic spirochetes, 368; Spirochzta recur- 
rentis, 368; Varieties, 369; Cultures, 369; Diagnosis of relapsing fever, 370; 
Spirocheta anserina, 371; Spirocheta gallinarum, 371; Spirocheta muris, 
371; Spirocheta icterohemorrhagie, 373; Spirocheta icteroides, 374; Fil- 
terability, 375; Yellow fever, 375; Transmission, 376; Prophylaxis, 376; 
Spirocheta hebdomadalis, 376; Spirocheta gallica, 376; Trench fever, 376; 
Transmission, 376; Spirocheta pallida, 377; Occurrence and morphology, 377; 
Cultures, 377; Luetin, 379; Syphilis, 380; Bacteriological diagnosis, 381; 
Microscopic detection of spirochétes, 381; Animal inoculation, 382 ; Wassermann 
reaction, 382; Luetin test, 386; Spirocheta refringens, 387; Spirocheta 
microdentium, 387. 


% 


TABLE OF CONTENTS xili 


CHAPTER XXVI. Tue FILTERABLE MICROBES 


The virus of foot-and-mouth disease, 388; The virus of bovine pleuro-pneumonia, 
_ 388; The virus of cattle plague, 388; The virus of rabies, 389; Occurrence and 
filtration, 389; Negri bodies, 389; Rabies, 390; Transmission, 390; Diagnosis, 
391; Pasteur treatment, 391; The virus of hog cholera, 392; Spirocheta suis, 
392; Immunity, 392; The virus of dengue fever, 393; The virus of phlebotomus 
fever, 393 ; The virus of poliomyelitis, 393 ; Occurrence and filtration, 393; Re- 
sistance, 393; Cultures, 393; Globose bodies of Flexner and Noguchi, 393; 
Transmission, 394; The virus of measles, 394; The virus of typhus fever, 394; 
The virus of small-pox, 395; Filtration, 395; Small-pox, 395; Vaccinia, 395; 
Immunity, 376; The virus of chicken sarcoma, 396. 


CHAPTER XXVII. MastIGOopHORA 


Herpetomonas musce, 397; Leptomonas culicis, 397; Cultures, 397; Trypano- 
soma rotatorium, 398; Trypanosoma lewisi, 400; Transmission, 400; Cultures, 
402; Pathogenesis, 402; Immunity, 403; Trypanosoma brucei, 403 ; Occurrence 
and morphology, 403; Transmission, 405; Cultures, 405; Nagana, 405; Diag- 
nosis, 406; Trypanosoma evansi, 406; Trypanosoma equiperdum, 406; Try- 
panosoma equinum, 407; Trypanosoma gambiense, 407; Occurrence and 
morphology, 407; Transmission, 407; Animal inoculation, 408; Human try- 
panosomasis, 408; Trypanosoma rhodesiense, 409; Trypanosoma avium, 
410; Occurrence, 410; Cultures, 411; Schizotrypanum cruzi, 411; Occurrence 
and morphology, 411; Animal inoculation, 413; Cultures, 413; Leishmania 
donovani, 413; Occurrence and morphology, 413; Cultures, 413; Transmission, 
413; Kala-azar, 415; Leishmania tropica, 415; Cultures, 415; Leishmania in- 
fantum, 416; Trypanoplasma borreli, 417; Bodo lacerte, 417; Trichomonas 
hominis, 419; Lamblia intestinalis, 419; Mastigamceba aspera, 419; Trimas- 
tigamoeba philippinensis, 419. 


CHAPTER XXVIII. RuHIzoPODA 


Ameeba proteus, 420; Occurrence and morphology, 420; Cultures, 421; Endameeba 
coli, 421; Occurrence and morphology, 421; Parasiticrelation, 422; Endamceba 
dysentericz, 423; Occurrence and morphology, 423; Relation of amebe to 
dysentery, 424; Cultures of dysenteric amebe, 425; Other rhizopoda, 426. 


CHAPTER XXIX. SpPoRozoa 


Cyclospora caryolytica, 42'7; Occurrence and morphology, 427; Pathogenesis, 429; 
Eimeria steida#, 429; Occurrence and morphology, 429; Sexual and asexual 
cycles, 429; Coccidiosis, 430; Eimeria schubergi, 431 ; Hemoproteus columbe, 
431; Occurrence and morphology, 431; Developmental cycle, 431; Hamopro- 
teus danilewskyi, 433; Fertilization in the sexual cycle, 433; Hemoproteus 
ziemanni, 434; Developmental stages, 434; Proteosoma precox, 436; Occur- 


xiv TABLE OF CONTENTS 


rence, 437; Cycle in the blood, 437; Sexual] cycle, 438; Plasmodium falciparum 
438; Morphology, 438; Sexual cycle, 440; Cultures, 442; Plasmodium, vivax, 
442; Cycle in the blood, 443; Sexual cycle, 443; Plasmodium malaria, 444; 
Developmental cycle, 444; Malaria, 444; Types of fever, 445; Diagnosis, 446; 
Mosquito carrier, 446; Prevention, 446; Plasmodium kochi, 448; Babesia 
bigemina, 448; Morphology, 448; Transmission, 448; Texas fever, 449; Babesia 
canis, 449; Gregarina blattarum, 449; Nosema bombycis, 449; Developmen- 
tal cycle, 450; Pébrine, 451. 


CHAPTER XXX. CILIOPHORA 


Paramecium caudatum, 452; Morphology, 452; Conjugation, 452; Opalina ran- 
arum, 454; Balantidium coli, 454; Parasitic relationships, 455; Balantidium 
minutum, 456; Spherophrya pusilla, 456. ; 


INDEXOOE-NAMES: 24 4a 4 YER BAERS RE BYERS ES awe « Abe 


LIST OF ILLUSTRATIONS 


Fie, Paar 
1. Image formation by means of a pin-point aperture..................4. 16 
2. Image formation by a single lens............ cc cece cece eee e ee aeee 17 
3. Image formation by two lenses in series, without magnification......... 17 
4. Image formation by two lenses in series, with magnification of two dia- 
TICLES iciie 4, seach Bek csinser eo Sectrsoweek, Beand Siavige es etareuntane. BANG Taye augue naan HEE 18 

5. Image formation by two lenses in series, with magnification of three 
diameters:. sss sexs aaa dawedies aaa Sane olde baes RySE Rte GUL EROS 18 
6. Microscope objectiveseissssciversssc tae ores Seee STAs MERE NEG deee teed 20 
7. Sectional view of compound microscope............0. 00. eeeee hana es 21 
8. Image formation in the compound microscope.............0e ec eee eaee 22 

9. Image formation in the compound microscope with an eye-piece of higher 
POWEr ssa 2s tea ey wees eduideed estas ceeds Aeearee erases ie 22 
to. Central illumination by the Abbé condenser............. Papeete Areal 24 
11. Illumination by a hollow cone of light................ 0. cece eee eee 24 
12. Illumination by a broad convergent beam?..............00..00 ee eeeee 24 
13: Dark-field condense: «30002 sss ences ta snes ees g ain iva see rea cho eens 25 
14. Optical parts of dark-field condenser........ 0.0... 00: cece eee eee aes 25 
1s. Production of the ‘‘dark outline picture”......... 0... c eee eee eae 26 
16. Production of the “bright outline picture”. ...... 0... eee eee 27 
17. Obliteration of outline by homogeneous illumination................... 27 
1B, MICHOSEOPE was asic idd salaiacein-digun SEES ORS et Atlas BGR SAR ME ea 29 
Tox Abbe Condens@E ss g..¢ceseaes eiweeea teases ane acaaen pec dau aoa 30 
vo. Platinund needlesic.dccccapaaeke peewaee a Gea Meee coe bated BOCAS 32 
ar. Pasteur pipettesi<sssiccae gees anoe sas cea sere aes aie apna wa emata datas 33 
22. Hanging-drop preparation......... Sere rn eee i Ra Cee SEES 34 
23. Cornet cover-glass forcepS........ 0. ccc eee eee t een eee nent e ees 38 
24. Stewart cover-glass forceps... 2.6.6... ccc cece eet cette eee e ee 38 
2s. Novy’s cover-glass forcepS......... 0.0.0 cece cece ence eens 38 
26. Kirkbride forceps for slides......... 0... c scene eee eee ene een ees 39 
27, MICTOtOMC es di seicdnee inte dee Dele MEE HME wg mE A Nudie Maree ea 57 
28. Hot-air sterilizer............... iihicfalethe seh Bibs Sane bis Ok cans Samet Hee 65 
29. Koch’s steam sterilizer... ........ 0 cece eee enn eee ences 66 
30. Diagram of the Arnold steam sterilizer........... 00.60 e sence eee ees 67 
31. Steam sterilizer, Massachusetts Board of Health.............-.-...04- 68 
32. Autoclave.............. ssa se arated orev ate atek aheniec ey yd apelin BR SENG Na duos ‘69 
33. Dissociation curves of indicators............ 6. se eee cece eee eee go 
34. Apparatus for filling test tubes.......... 6.0.0 cece ee cence eens gI 


XV 


LIST OF ILLUSTRATIONS 


Fic. PAGE 
36: Potato: tube sys cscs seag udu ghee se Rewer see A wow ee ne eden Hamers oe 93 
36. Koch’s serum sterilizer......... 0.0 e eee eee eee tenes eAMRe 096 
37. Pipette for collection of sterile blood,.......-.-- ++ eee eee ee eee eens 98 
38. Pipette for collection of sterile blood from an animal.............. Sesve 90 
39- Taylor’s tube for vein puncture......... 660 e eee eee ee eee 105 
40. Instrument for collection of feces from infants.............0. eee ee eee 106 
41. Method of inoculating culture media............. 00 cc cece eee III 
POs POU LIS Hiss aed judas a: cavnseasa laces mreaene isn) ae E ROB RUAN SBE STORE ONE 112 
43. Colonies in gelatin plate.......... 0.6 c ce eee nents 113 
44. Manner of making Esmarch roll-tube...... Che al ale Biya aracalcy oe en ave AN his II4 
45. Dilution cultures in Esmarch roll-tubes............. 00000 e eee eee es 115 
46. Stab-culture closed with rubber stopper...........0. 000000 c eee e eee ee 118 
47. Smear culture closed with rubber cap.........0 0. sce c eee e eee eens 118 
48. Uncubator ye cccwerwk he eeoees eee ner whine need pre em ans 120 
49. Reichert gas-regulator........... 0.00: eee ee eee eee eis TIME RATE 121 
50. MacNeal gas-régulators occa suns cicsanes eewee eins bie Hees eee wee ea 122 
51. Roux bimetallic gas-regulator............0 00. c cece eee eee + 123 
52. Koch automatic gas-burner.............0 eee eee Druid Gaaeningie Pavers 123 
53. Diagram of electric regulator for low-temperature incubator............ 126 
54. Aerobic culture by Buchner’s method ............ 0.0: c cece eee cease 130 
55. Novy anaerobe jar for tube cultures.......... ee rn eee 131 
56. Novy anaerobe jar for Petri dishes or tubes................ 00. ceeeees 131 
57. Novy anaerobe jar, improved pattern............ 0. cece cece e eee eens 131 
58. Tripod and siphon flask for anaerobic culture by combined hydrogen and 

pyrogallatemethod srr. ssemadiuls Seaweeds see wesandacae’ teased 132 
59. Aerobic organism that will not grow under a cover-glass................ 133 
60. Method of making collodion capsules.............0 ccc eeeeeeeeeucees 139 
Gro Commonsnolds) cis ancnaweadaseana toawos Saawowns Gealnn Ga es 144 
62. Yeast cells stained with fuchsin..............00. 000.0 ccc cence nena 145 
63s. Wine and! beer yeasts cars xis saves oy sundcn dies sonsseisctnred-g Sask dmb eehardh scenery 3B 146 
64. Various groupings of micrococci............. 00000 cece eee e ea sues 148 
65. Bacilli of various forms 6 cciccies awa hauhan dene naa vies eed Mowers ee eas 151 
66. Sporulation...... F falar st ante, Daan at AVM Dp AS ate aang @ Gaeta ap SHE Gite ATS bs Anew Abbe a I51 
67. Various positions of sporeS.......... 0.00. c cece cece cece ceeeeeevetens 152 
68. Types of spirilla ee Te se Miasacned Asnicneaae edd tauaonavalsheeaasen 153 
69. Bacteria with capsules.........0.. 00.00 cc cece cece cece ee vecenneeuuns 154 
7o. Bacteria showing flagella..... 0.0.0.0... e ce eeeeeees 154 
vr: Formation Of Spores.c. ssciess nanan neees sos own eRe rade badeegure aaaes 155 
72. Bacteria With Spores) sox a4dee cmavaea canals oe aieaw abe es bose di de ees 155 
#3. GetMination OF SPOLresés wae sad veies eax cam y ¥esobeesinc ae sonia Raed oma ea Law leaee 155 
74. The most important trypanosomes...........0.0. 0c. cece ce eeceecuee 158 
75. Leishmania donovani. . 0... 6c cece ccc cece ee eceues 158 
76. Leishmania donovani in culture.......... 0.00.00 e ccc cece cece eves 159 
77. Trichomonas hominis......0 00.0. i ici e eee e eee eee ceed EBQ 


Fic. PAGE 
78. Lamblia intestinalis... 000.000 n ccc ee eeeeuceus 159 
79. Endameba coli ..... 6. ccc ccc c eet eeetceteeenes 160 
80. Developmental cycle of Eimeria schubergi....... ova Renee's cates efeeen nae are 162 
8r. Asexual cycle of Plasmodium falciparum. ....00.0 00.0 c cece cee eens 163 
82. Forms of Babesia muris....000 0000 e eee eeeneeens 164 
83. Developmental cycle of Nosema bombycis........ 00. c ccc ccc cen ee aes 165 
84. Sedgwick-Tucker aerobioscope..........6.000 0000 cece cece neue veunenees 184 
85. Jeffer’s plate for counting colonies...............00 00 0c cece cece ee eens IQI 
86. Surface divided into square centimeters for counting colonies.......... 192 
87. Receptor of the first order uniting with toxin......................00. 216 
88. Receptor of the second order..........0 0.0... ccc cece eee eeneeecceuaes 217 
89. Receptor of the third order.......... 00... ccc cece cece nee e ees 221 
go. Deviation of complement.............. 0... cece cece enn nena 223 
QU WEUCOR IN UCM Oe scaceinie o-5 sig as hee saep creck aoe BoE ign SEN Rg HONG NMEA Ce ans 238 
OD. MUCOE CORVMOLL OR sects erica’ ion agnor ae SRR a ean NAA 2 oe AN £8 be pe a a ie 238 
93. Aspergillus glaucus.......0 00.0 ccc ccc ee eee ens ipeaeas steams 230 
94. Penicillium crustaceum......00000 00 ccc ccc cece een eeeees 240 - 
95. Coccidioides immilis....... 0 occ ccc cece e eee ees fae hee 242 
G0: O10 cm LACS aie etis eienndg Sons cea phy Se yas Causes gana eceusaedes 244 
97- Oidium albicans, colony... 6.6.0... ce eee eee teen ees 245 
98. Oidium albicans, mycelial thread.............0 000. c cece ees 246 
99. Scutulum of favus on the arm of aman............0.00 00 cee cence eens 247 

roo. Scutulum of favus in a mouse:.......... 0.00 c eee eee 248 

tor. Achorion schoenleinii, colony.............0. 0c cece cect eee tans 249 

102. Sporotrichum schencki, cultures on agar... 1.1... cee 250 

103. Sporotrichum schencki, forms of mycelium............... 00000 e eee eee 251 

ro4. Organisms found in oidiomycosis............ 0.0.0 eee eee eee eee 252 

LOS: AClNOMNCES DOvtS cosas ened score tae peek eS tea nines we ey Aa a ae 255 

ro6. Gonococci and pus cells............ cee eee es 259 

107. Meningococcus in spinal fluid....... 0... cece ene 265 

108. Pneumococcus showing capsule........... 0.6006 e cece cee ee eee 267 

tog. Staphylococcus aureus, gelatin culture... ........... Bh ered nants Sema ane 276 

£105 BOCtUS SUDUGLIS is sais aie acd wiaease a Goad oe Rage we eae When wen OSTA S 280 

111. Anthrax bacilli in capillaries of the liver.........0. 0.0... cece seen ees 281 

112. Bacterium anthracis showing SPores............60..0 eee ee edn eee eens 282 

113. Bacterium anthracis, colony upon a gelatin plate..............+.....00- 282 

114. Bacterium anthracis, thread formation of colony.................000005 283 

115. Clostridium perfringens, in agar showing gas formation................. 288 

116. Tetanus bacilli, showing terminal spores.......... 00.0000 cece eee eens 292 

117. Clostridium tetani, stab culture..........0.0 00sec eee eee teen eens 293 

118. Clostridium botulinum......... Sodding. hilaun iSite had Raabe CEA EOSS TEE 204 

tig. Bacillus of diphtheria ......... 0... ccc ce eee ents 299 

120. Bacillus diphtherie stained by Neisser’s method................- ee 299 

121. Forms of Bacillus diphtheria in cultures on Loeffler’s serum............. 300 


LIST OF ILLUSTRATIONS Xvii 


xviii LIST OF ILLUSTRATIONS 

Fic. Pace 
122. Forms of Bacillus diphtheri@ on agar............ ia eaiasaNann 2 eiepans umes 300 
123. Colonies of Bacillus diphiheri@ on glycerin agar..............0 000 e eee 301 
124. B. diphtheriew, culture on glycerin agar...........6. 00.0 cece eee eee 302 
125. Swab and culture tube for diagnosis of diphtheria..................... 303 
126. The Morax-Axenfeld bacillus.......... 0... ccc cece eee eee ene 310 
127. The Koch-Weeéks bacilluis. oaiscscsso eg cong eon ver aind ve amciuen enema ae 310 
128. Bacillus tuberculosis in sputum............. GY MRE ES ROME aE EN 314 
129. Bacillus tuberculosis from a pure culture.......... 0... cece eee ee 315 

130. Tubercle bacillus showing branching and involution forms..... Erepensiseanes 316 
131. Bacillus tuberculosis, culture on glycerin agar...........0.. 00 cece ee eee 317 
132. Bacillus of bubonic plague....... Aine i OhewUd TY ERR Ee ERY eT < sara 330 
133. Bacillus coli, showing flagella........00.0. 00 ccc cece cece teens 337 
134. Bacillus coli, superficial colony on gelatin plate....................0005 338 
135. Friedlander’s pneumobacillus, gelatin stab-culture....................+ 340 
136. Bacillus of typhoid fever... ccc c ewer ees vegan t doutesi es vanes 343 

137. Bacillus typhosus, showing flagella...........0. 0.000 c cece cee cee ++ 344 
138. Colonies of Bacillus typhosus and Bacillus colt.........0 0000 cece ec ees 344 
TOS BOCES MOLL CG are ws a caceu mare 9 open eite 8 8 ume metre B wbe bean el ones wea alee ans 354. 
140. Cholera vibrios, short form. ..........0 0.00000 ccc cece eee eens aee 361 

141. Cholera vibrios, showing flagella............0.0. 0.0 0e ccc e cece eee ee eeeee 362 

142. Involution forms of the spirillum of cholera................... 0000000 362 

143. Spirochsete of relapsing fever........ Nps bet Capi eesre Alec ee tun ees gaint Ne ves 369 

144. Spirocheta recurrentis in blood of arat.......0.0.0.. 00 cece eee e cece ees 370 
145. Spirocheta (morsus) muris in MOuse..........0. 000 cece cece cee eee 372 

146. Spirocheta (morsus) muris in guinea pig....... 2.0.0.0. 00.0 ccc cece eee 372 

147. Leptospira icterohemorrhagi@.......0..600 0c ccc cece nee cn eees 373 

148. Leptospira icteroides in blood..........0.0..0 ccc eee cence eeu eeunaeens 374 

149. Leptospira icteroides in culture.......... 0.000. ce ec eee cceeeeunaes 374 

150. Aédes (slegomyia) calOpus .... 66... ccc cece cen ecneueevecs 375 

151. Preparation showing Spirocheta pallida and Spirocheta refringens........ 378 

152. Spirochata pallida stained by Levaditi method............ stadia kumameacsne: BOO! 

153. Negri bodies in brain of a rabid dog........ 0.0.00 cece eccucececeeuaes 390 

154. Herpetomonds MUsC@..... 0.6 cece cnc ccc ecuceucenevus 307 

155: Leplomomas culi6ts. ...ccwcuncccuuws uns eaeun dn ivauarevsiaensasaunas 398 

156. Trypanosoma rotatorium in blood of a frog..........00. ccc cece ee veves 399 

157. Trypanosoma rotatorium in culture......... 00.000 ccc ccc cece cceceues 399 

158. Trypanosoma lewisi..... 00. ccc ccc ccc cece nececntveveenes 400 

159. Trypanosoma lewisi, various multiplication forms.................0.000: 401 

160. Trypanosoma lewisi, eight-cell rosette... ........ 000. ccc cece cee eceee 402 

161. The most important trypanosomes parasitic in vertebrates.............. 403 

162. Glossina morsitans, dorsal view..........0 00 0c cc cee cccceeccucuccucues 404 
163. Glossina morsitans, lateral view........0.. 0.00 c cece cece cccccucucs 404 
164. Trypanosoma equiperdum...... 0.0600 0c cece cece cee ee By seen Qian 406 

165. Glossina palpalis....... ig sdetails ns avid abnbtneed vsti 408 


LIST OF ILLUSTRATIONS xix 


Fic. PAGE 
166. Trypanosoma avium in blood of birds...... oi gue Aglaabececsnad eeeacn ees 409 
167. Trypanosoma avium in culture... .... 06. nett eee nee 410 
168. Schizoirypanum cruzi in tissues of the guinea pig....................45 412 
169. Schizotrypanum cruzi in human blood......... SAIS a eMatraca arkacionun ye aehsy 413 
170. Conorhinus megistus .. 0.0.06. e nett eenes 414 
171. Leishmania doncvani in spleen juice ............00 060.0 cece eee 414 
172. Leishmania donovani in culture......... 0.000 cee eee 415 
173. Leishmania tropica in pus........ 06. eee eee een nee 415 
174. Leishmania tropica in culture........0.. 0000 cece eee 416 
175. Trypanoplasma CYPrini.... 6. cette ee peees 416 
176. Bodo lacert@....... 0.6 ttc eens eee rere 417 
177. Trichomonas hominis...............00 000 ccc eens 417 
178. Lamblia intestinalis... 00 ce nee . 418 
179. Trimastigameba philippinensis ...0. 00000 ee 418 
180. Ameba proteus 00. eee eee t teen nee 420 
Stas Se 2/2700 1A :) oe ee 421 
182. Endameba dysenterie, unstained ..............0 0000 ees 423 
183. Endameba dysenterie, stained preparation.................0..0 eens 423 
184. Endameba dysenteri@, mature cyst............ 2s 424 
185. Cyclospora caryolytica, male cells............0. 00.0 ccc cee eee 427 
186. Cyclospora caryolytica, female cells.............0. 00 cece ee 428 
187. Cyclospora caryolytica, fertilization and production of sporozoits......... 428 
188. Exmeria steid@, OOCYSt...... 6. eee eee ee sa ate 420 
189. Eimeria steid@, various forms ........... 0.0... eee ees 430 
190. Hemoproteus columbe, developmental cycle..............0 00.0 eee eee 432 
191. Hemoproteus danilewskyi..... oleate Cchntade citar ele aaa ceee eon oie cient: Sicitagin ABS 
192. Hemoproteus ziemanni, gametocytes........... Jugeedede Seen aes: aveed 434 
193. Hemoproteus ziemanni, formation of microgametes and fertilization...... 434 
194. Hemoproteus ziemanni, various forms observed in blood................ 435 
195. Developmental cycle of Proteosomd......... 0666 ccc ccc eee 436 
196.'Proteosoma precox in blood of a lark... eee eee 437 
197. Midgut of a mosquito showing odcysts.of Proteosoma........... 6000605 437 
198; OGCyst OF PrOleosomess vice paia iy <quga a aies MEMO E eed tw O45 dee eee RHI 438 
199. Plasmodium falciparum, various forms observed in the blood............ 439 
200. Capillary of brain filled with Plasmodium falciparwm............00.0065 439 
201. Plasmodium falciparum, development of the gametocytes............... 440 
202. Stomach wall of Anopheles infected with Plasmodium falciparum........ 440 
203. Digestive tract of Anopheles, infected with Plasmodium falciparum....... 441 
204. Plasmodium falciparum, ripe sporozoits in odcyst......... 666. c eee eee 441 
205. Salivary gland of Anopheles, containing sporozoits of Plasmodium falci- 
POTUNE asta ss. isre aieastatec bape 18 alaus eared a:b BURT a Gea REA ee id DREN a I en, os Bue 442 
206. Plasmodium vivax, stages in asexual cycle................ Gb bee Bemadamin 443 
207. Plasmodium vivax, sporulation .........0.. 000 c ccc eee eens 443 
208. Plasmodium vivax, double infection .........0.... 06. c cee 443 


LIST OF ILLUSTRATIONS 


. Plasmodium vivax, stages in development of the gametocytes............ 
. Plasmodium malaria, asexual cycle..........66 6.66 c cee ete eee 
. Plasmodium malaria, gametocytes..........6 06 0c ccc ee 
. Comparison of Culex and Anopheles......... 00060 c cece eens 
ss SED OSTD: ORE MIANG hier wr AB echoes cael y Wego oe Mania Nish BADR WRAL sett 
 GRORAPING DIGUET Ut ecco Fed Hid eeoed FACE RE RS ROK BRSEES HERS DOR OOS 
sy VOSCmG DOMDIN ETS te os Bea ev xx a aesacectvn ies iapigus RASS ah Gebla tO Le SUE vl AERE 
» Paramecium: coud alts ss ise ccc iccouu une ooea es Eka REEL EE Le ede’ 
. Paramecium caudatum and Paramecium putrinum..... 0.6000 ccc ee 
AO POMC FANON aie tN aes ct rela SE Re an la reads ee Le a rears 
BUCA COltroscctirs cieeinde s Seseaen es Ma dE cag Ha eeMSAR Ee Syed 


INTRODUCTION 


Bacteriology and Microbiology.—The science of Bacteriology 
occupies a somewhat peculiar position among the natural sciences, 
partly because of its recent development and partly because of the 
overshadowing importance of its practical applications. As, 
bacteria are microscopic plants, some have considered bacteriology 
as a minor division of botany; but the methods of work and the 
practical applications of bacteriology have little in common with 
those of the more ancient science. Indeed were it not for the 
‘importance of these little organisms to the chemist, the pathol- 
ogist, the physician and the agriculturist, we should hear little 
about them. 

The foundations of the science were laid by Pasteur (1858) 
by the introduction of media and methods for artificial culture 
of bacteria and the separation of mixtures into pure culture by 
the laborious and uncertain but nevertheless successful method 
of dilution in fluid media, thus making possible the accurate 
experimental study of microbes. Robert Koch (1872-1882) con- 
tributed much to the establishment of the new science by intro- 
ducing the use of solid media and the method of plating for the 
isolation of pure cultures and especially by his wonderful achieve- 
ments in investigation of the pathogenic bacteria by his new 
methods. Koch used potatoes, and aqueous humor and blood 
serum rendered solid by the addition of gelatin. He first em- 
ployed the anilin dyes in staining bacteria (1877), microphotog- 
raphy of bacteria (1877), homogeneous immersion objectives 
and the Abbé illuminating apparatus (1878). Much of our 
modern technic has been devised by his pupils and colleagues. 
The commonly used meat-water-pepton-gelatin was introduced 
by Léffler; agar by Frau Hesse. 


I 


2 INTRODUCTION 


The development of bacteriology has been promoted by the 
work of biologists, botanists, chemists, pathologists and agrono- 
mists, many of whom have been willing to include bacteriology 
as a subdivision of their own field. The practical importance of 
bacteriology to these various fields is becoming progressively © 
more evident. The relation to pathology and medicine is per- 
haps most clearly recognized, although the importance of bac- 
teria in chemical technology and in agriculture is no longer 
questioned. The relationships to general biology have not been 
so completely developed as yet, partly because these have seemed 
to offer less promise of immediate practical application, and partly 
because few well-trained zoologists or botanists have devoted 
serious attention to bacteriology. 

As a matter of fact, bacteriology must be ranked as a distinct 
science, especially because of its peculiar technic and because 
of the peculiarly critical thought necessary in the interpretation 
of bacteriological observations and experiments. The importance 
of these can be fully appreciated only after actual experience 
in handling microbes. Here is a science in which skepticism 
is a necessary safeguard, a skepticism which will become con- 
vinced only when overwhelming evidence compels conviction; 
and while regarding other conclusions with interest or even with 
enthusiasm, still carefully reserves final judgment as long as the 
observed phenomena are open to more than one interpretation. 

These methods of thinking and of working have been applied 
to organisms other than the bacteria, on the one hand to the 
unicellular animals, the protozoa, on the other to more complex 
plant-forms such as the yeasts and molds, and more especially to 
the study of the still undefined types of living things known as 
filterable viruses or more vulgarly as the ultramicroscopic mi- 
crobes. Inasmuch as many of these live as parasites and some 
are important in the causation of disease, they are commonly 
considered along with the pathogenic bacteria. The terms mi- 
crobe and micro-organism properly include these as well as the 
bacteria. There is thus an-evident tendency to extend the field 


INTRODUCTION 3 


of bacteriology so that it becomes microbiology or the science of 
micro-organisms. There are many reasons why this is desir- 
able. It is certainly essential that the microbes included among 
the protozoa and the filterable viruses should receive more atten- 
tion in the future, both from beginning students and from trained 
investigators. Until separate instruction in these subjects is pro- 
vided for medical students, they may perhaps best be studied 
along with bacteriology. 

Biological Relationships.—Since ‘ie earliest times, the essen- 
tial difference between living things and lifeless things, that is, 
the nature of life, has been an interesting subject of speculation. 
It was at first assumed as a matter of course that the transition 
from lifeless to living matter readily took place without the 
agency of preéxisting living matter. This speculative assump- 
tion is still not without its able supporters. The history of actual 
observations, however, is one long record of refutation of this 
assumption wherever the facts have been subjected to accurate 
observation. The ancient Greeks held that living beings arose 
spontaneously and even Aristotle (384 B.C.) asserted that ani- 
mals were sometimes formed in this way. These ideas were dis- 
proved by more careful observation. A notable experiment was 
that of Francesco Redi (about 1650) who allowed meat to putrefy 
in a jar covered with fine wire gauze. The flies attracted by the 
odor deposited their eggs on the gauze and the maggots were 
hatched there. The assumption that the maggots arose de novo 
in putrefying meat was thus disproven. Harvey in 1650 made the 
famous statement, ‘‘Omne animal ex ovo’ which was later ex- 
tended to ““Omne vivum ex vivo.” 

When Anthony van Leeuwenhoek, the “Father of micro- 
scopy,” discovered, described and figured bacteria in 1683, the 
assumption of spontaneous generation was at once applied to this 
group of organisms and, although rendered exceedingly doubtful 
by the experiments of Spallanzani (1777) and of Schulze (1836), 
it still continued to be accepted by many scientific men until it 
was combated by Pasteur, 1860 to 1872. ‘After the accurate 


4 INTRODUCTION r 


observations of Pasteur upon fermentation and putrefaction and 
his successful defense of them through a long period of contro- 
versy, the assumption of spontaneous generation as applied to 
bacteria was discredited and has been very generally given up. 
“Only a very few observers! still claim the existence of evidence 
in support of its application here. The more prominent advo- 
cates? of the assumption of spontaneous generation or abio- 
genesis seem inclined now to apply it to some group of living 
beings still beyond the limits of actual observation. 

Closely related to the assumption of abiogenesis has been the 
assumption of heterogenesis among the bacteria, the notion that 
various kinds of microbes could readily be produced from one 
species. Although very successfully combated by Pasteur, 
this idea still persisted for many years in the early bacteriological 
literature, the observed new species of microbes actually resulting 
from faulty technic by which new germs had gained entrance to a 
previously pure culture. These observations are often repeated 
unwittingly by -beginners in bacteriology. The validity of bac- 
terial species is now unquestioned. On the other hand, the vari- 
ability in the descendants of a single cell through a greater or 
less range, and the possibility of producing morphologically and 
physiologically different strains of the same species by appro- 
priate environmental conditions are now well known, resulting 
again very largely from the pioneer work of Pasteur in the produc- 
tion of attenuated cultures of the germs of chicken cholera and 
of anthrax. 

The systematic relationships and the classification of bacteria 
were first studied by O. F. Mueller (1786). Ehrenberg (1838) 
made the first serious attempt at a comprehensive classification 
and many modern systematists are inclined to return to his work 
to establish authoritative terminology for present use. He re- 


? Bastian, The evolution of Life, London, 1907. The Origin of Life, London, 
1013. 

® Schafer, Nature, Origin and Maintenance of Life, Science, 1912, Vol. XXXVI, 
Pp. 289-312. 


INTRODUCTION 5 


garded the bacteria as animals. Ferdinand Cohn (1872) recog- 
nized the nature of bacterial spores, showed the close relationship 
of bacteria to the alge and established their classification in the 
plant kingdom. He distinguished six genera—micrococcus, 
bacterium, bacillus, vibrio, spirillum and spirocheta. Migula 
(1897) undertook an extensive revision of bacteriological nomen- 
clature and classification, basing it upon morphological characters, 
and his- system is doubtless the most satisfactory yet offered. 
The subject is still in a very unsettled state, nevertheless, and 
there is no system of classification generally accepted by bac- 
teriologists. The problem presents so many ‘difficulties and our 
knowledge of the bacteria is still so incomplete that many authori- 
ties seem prone to consign systematic classification to the future, 
and to employ names of sufficient historical prominence to insure 
their correct interpretation. 

Fermentation and Putrefaction—The relation of micro- 
organisms to the decomposition of organic matter, fermentation 
and putrefaction, was one of the first fields of applied bacteri- 
ology to be studied. Following the observation of bacteria in 
saliva by van Leeuwenhoek in 1683, micro-organisms were dis- 
covered in all sorts of decomposing material. At first, these or- 
ganisms were regarded as unimportant for the chemical process 
and interest attached chiefly to the question of their origin, 
whether by spontaneous generation or from previously living 
cells. Needham (1745) directing his attention more particularly 
to this first question, boiled an infusion of meat and, keeping it free 
from contact with the air, nevertheless observed after some days 
the presence of ‘‘infusoria.’’ Spallanzani (1765) repeated Need- 
ham’s experiments, subjecting hermetically sealed flasks of meat 
infusion to the temperature of boiling water for one hour, and 
he found no subsequent development of life and no decomposition 
of the infusion as long as it remained sealed. While discussion 
continued concerning the discrepancy between the results of 
Needham and Spallanzani and concerning the relation which 
the subsequent exclusion of the air might bear to the absence 


6 INTRODUCTION 


of life in the flasks, the method of heating was applied to the 
preservation of vinegar by Scheele (1782) and to the preser- 
vation of foods in general by Appert (1811). The method was 
quickly introduced into other countries and developed by various 
tradesmen, who attempted with more or less success to keep their 
processes secret. Success in preservation by canning remained 
somewhat uncertain, as a precise understanding of the underlying 
scientific principles was still lacking. Schulze (1836) showed that 
air might be admitted to flasks prepared by Spallanzani’s method, 
without the development of life and without putrefaction, pro- 
vided the air were first passed through a series of bulbs containing 
concentrated sulphuric acid. The subsequent work of Schréder 
and van Dusch (1853), who obtained similar success by filtering 
the air through cotton, of Pasteurand Tyndall (1860-62) , who were 
-able to preserve putrescible fluids directly in contact with air, 
provided the air were rendered perfectly free from dust, has 
established the fact. that the decomposition ordinarily taking 
place after exposure to the air is due to the introduction of living 
germs into the previously sterile material. 

The idea that specific kinds of fermentation are caused by 
specific kinds of microbes was first clearly put forward by Schwann 
and Cagniard-Latour (1837), who showed that yeast-cells were 
living organisms and claimed that the alcoholic fermentation of 
sugar solutions was due to their growth. The importance of this 
relationship received little recognition until Pasteur (1860-72), 
during his extensive and careful researches into the nature of 
fermentation and the causation of undesirable fermentation (dis- 
eases of wines and beers), demonstrated conclusively that the 
kind of decomposition of a fermentable substance depended upon 
the nature of the substance, the kind of microbes present and the 
environmental conditions, such as temperature and presence or 
exclusion of air. The mere introduction of a small number of 
unfavorable microbes was sufficient to change the whole nature 
and course of the fermentation. Furthermore, Van der Brock 
(1857) and Pasteur (1863) were able to collect such fermentable 


INTRODUCTION 7 


materials as grape juice, wine, blood, tissues of plants and animals 
and preserve them free from decomposition and from all microbic 
life, merely by effectively avoiding contact with germs during 
collection and storage. 

The agency of microbes in fermentation was ridiculed by 
Liebig, the most prominent chemist of the time, who steadfastly 
continued to regard decomposition of organic material as a 
purely chemical ‘process uninfluenced by biological activity. His 
ideas prevailed for a time because of his prominent position. 
The correctness of Pasteur’s contention is now universally ac- 
cepted. Nevertheless it should not be forgotten that many 
organic substances are in themselves so unstable that even in the 
absence of microbic life they disintegrate, or become oxidized in 
the presence of the air. These changes are different from those 
ordinarily known as fermentation and putrefaction. 

Pathology and Hygiene.—The history of the development of 
our ideas concerning the relation between microbes and disease 
is one of the most interesting and perhaps the most important 
chapter in the history of bacteriology. The customs and rec- 
ords of the ancients give evidence that they recognized the pres- 
ence of an unseen agency in the body of the diseased individual 
capable of causing sickness in others. This was recognized by 
the ancient Persians as recorded by Herodotus. The isolation 
of lepers by the ancient Hebrews shows that the infectious char- 
acter of this: disease has long been known, though other affec- 
tions than leprosy were probably confused with this disease. ‘He 
is unclean; he shall dwell alone; without the camp shall his 
habitation be.’’ (Lev. XIII, 46.) There is, in fact, much in the 
laws of Moses that points to some knowledge of the nature of 
infection. ‘This is the law, when a man dieth in a tent all that 
come into the tent and all that is in the tent shall be unclean for 
seven days. And every open vessel that has no covering on it 
shall be unclean.” (Numb. X1IX,14,15.) “Everything that may 
abide the fire, ye shall make it go through the fire, and it shall 


8 INTRODUCTION 


be clean.”” (Numb. XXXI, 23.) In Homer we read of Ulysses, 
that, having slain his wife’s troublesome suitors: 


“With fire and sulphur, cure of noxious fumes, ‘ 
He purged the walls and blood-polluted rooms.”’ (Pope’s Odyssey.) 


These records certainly suggest a rather advanced state_of knowl- 
edge concerning the nature of contagion. It may be that they 
record customs derived from a superior knowledge of some other 
ancient people, perhaps the ancient Egyptians. During the 
middle ages, as doubtless also before the dawn of history, epi- 
demic disease was regarded as a visitation of Providence or at- 
tributed to the influence of gods, demons or other supernatural 
agencies. Epidemics were associated with the appearance of | 
comets in the sky or with other evidences of divine wrath. These 
conceptions of disease have not altogether disappeared even at 
the present time. 

Hippocrates (400 B. C.) denied the supernatural causation 
of disease and held that such doctrines were mere cloaks for help- 
less ignorance. He ascribed epidemic disease to a morbid secre- 
tion of the atmosphere, and later writers have expressed this 
idea of a morbid secretion by the word miasm, its exact nature 
remaining for centuries intangible and mysterious. There is 
here a conception different from that upon which the hygienic 
measures of the Persians and Hebrews were founded and the 
distinction was clearly expressed by Pettenkofer in the nineteenth 
century, who defined contagious diseases as those which are trans- 
mitted directly from man to man or through the agency of solid 
objects, while in miasmatic diseases the causative agent enters 
from the outside world where it may live naturally or where it 
must have undergone a ripening process since its escape from the 
body of the sick person. As will be seen later these ideas apply 
very well to certain diseases, for example, small-pox and syphilis 
as contagious diseases and yellow-fever and malaria as a mias- 
matic. The ancient Greeks recognized the contagiousness of 
several diseases and Galen classed plague, itch, ophthalmia, con- 


~ 


INTRODUCTION 9 


sumption and rabies as contagious. Fracastorius (1546) during 
the period of the great epidemic of syphilis in Europe, published 
a book containing the first comprehensive discussion of the theory 
of contagion. He recognized contagion by contact, by fomites 
and at a distance. Soiled material of all kinds was included 
under fomites, as also those healthy individuals capable of trans- 
mitting disease, a phenomenon already recognized. Transmis- 
sion by insects and animals was also included under this head. 
The transmission ‘‘per distans’ was considered due to emanations 
from the patient diffusing to a distance through the atmosphere. 

Kircher in 1658 claimed to have seen the living contagium in 
the body in the form of minute worms, and his observations were 
widely recognized. The objects he saw were not accurately 
described but it seems very certain that they were not bacteria. 
Probably they were the normal cells of the tissues. 

The discovery of bacteria by van Leeuwenhoek (1683) was 
not immediately recognized as of importance for the germ theory. 
Leeuwenhoek himself considered it impossible for his ‘‘animalcula”’ 
to penetrate into the blood because of the compactness of the 
epithelial tissues. 

Almost a century_later, Plenciz (1762) maintained that each 
infectious disease must have its own specific cause. Reimarus 
(1794) also expressed the same opinion and considered these living 
organisms to be of the order of infusoria or perhaps still smaller 
beings not yet visible with the microscope. These ideas were 
not supported by objective evidence and received only passing 
attention. They were soon thrust aside by other interesting if 
less valuable speculations. 

The development of general knowledge of the animalcules in 
the early part of the nineteenth century, already referred to 
in the discussion of the biological relationships and of fermenta- 
tion, was preparing the way for progress in the problem of disease. 
In 1834 the contagium vivum of itch, the itch mite (Sarcoptes 
scabei), a fairly large mite to be sure, was rediscovered and its 
- relation to the disease made evident. In 1837, the same year 


To INTRODUCTION 


in which Cagniard-Latour. and Schwann established the relation 
of living yeast to alcoholic fermentation, Donné described vibri- 
ones (bacteria) in syphilitic ulcers, and Audouin amplified the 
discovery of Bassis that muscardine, a disease of the silkworm, 
was caused by a mold (Botrytis bassiana) which was transmitted 
from the sick to the healthy worms by contact or by air currents. 
These discoveries furnished a great impulse to further in- 
vestigation. 

Henle (1840) reviewed the evidence then at hand and 
concluded in a very logical way that the causes of contagious dis- 
eases were to be sought for among the minute living micro-organ- 
isms. He recognized that no human disease had yet been shown 
to be caused by a micro-organism and he formulated the require- 
ments to be fulfilled in order to prove such a relation, namely, 
that the microbe must be constantly present_in the disease, 
must be isolated from the infectious material, and must then 
alone be capable of producing the disease. 

During the next twenty years, the attempts to discover the 
cause of an infectious disease and to satisfy the postulates of 
Henle were successful in several diseases due to molds, Favus 
(Achorion Schoenleinii) 1839, similar skin diseases known as 
trichophytosis and pityriasis and especially thrush, shown to be 
caused by Oidium albicans by Robin in 1847; but in all the more 
important diseases only failure resulted. The reawakened interest 
in contagium vivum therefore again gradually faded away. 

During this time Pollender and Davaine and Rayer (1850) 
had discovered the minute rods in the blood of animals sick with 
anthrax, and in 1863 Davaine had proved the almost constant 
presence of these rods in the disease and the possibility of trans- 
mission by inoculation from one animal to another. 

Pasteur from 1865 to 1868 investigated the fatal disease of 
silk-worms known as pébrine, discovered the microsporidium 
(Nosema bombycis) which occurs in the sick worms and in the 
eggs, and devised a successful method of eradicating the disease. 

In 1870-71 the presence of bacteria in wounds and in the 


INTRODUCTION II 


internal purulent collections in pyemia and septicemia was first 
definitely recognized by Rindfleisch (1870), but more especially 
by Klebs in a large number of cases at the military hospital at 
Karlsruhe. The latter observed spherical bacteria arranged in 
groups or as a rosary, to which he gave the name Microsporon 
septicum. His observations were quickly confirmed by other 
competent pathologists. Similar organisms were soon found 
in a great many wounds and other inflammatory processes. 
Specific causal relationship was still unproven. 

In 1873 Obermeier described the slender but actively motile 
spirochetes seen by him in the blood in relapsing fever as early 
as 1868. 

In 1874 Billroth concluded that there was still no disease in 
which the causal relationship of micro-organisms had been con- 
clusively proven. The skin diseases due to molds were relatively 
unimportant and had not been recently studied. The microbes 
found in other diseases might just as reasonably be regarded as 
a product of the disease or as only incidental to it. Even in 
anthrax, where the evidence seemed strongest, there were cases 
of the disease without the presence of the peculiar rod-like bodies 
in’the blood, and indeed these rods might be crystals and not 
living organisms at all. 

Since 1867 Lister, stimulated by the investigations of Pasteur 
on fermentation and putrefaction, had been developing and 
applying an antiseptic method to the treatment of wounds, 
which consisted of the use of carbolic acid. The results of this 
method published in 1875 were so remarkably favorable that it 
was quickly adopted throughout the world, and its success did 
much to prepare the way for the recognition of the réle of microbes 
in suppuration, if it did not in itself convince. 

Robert Koch, 1876-1881, first satisfied the postulates laid 
down by Henle, and again formulated by himself, in the bacterial 
disease, Anthrax. The presence of the bacilli in the blood of 
animals suffering from anthrax had been established by a large 
number of previous workers, and the transmissibility of the dis- 


12 INTRODUCTION 


ease by inoculation with blood of diseased animals was already 
known. Koch was able to grow the bacillus in pure culture in a 
test tube, using the aqueous humor of the ox’s eye as a medium. 
He was able to observe growth and division and the formation 
and germination of spores under the microscope. Finally with 
these cultures, which had been propagated a long time in the 
culture medium, he was able again to cause anthrax by injecting 
them into susceptible animals. The demonstration of the causa- 
tion of disease by bacteria had been achieved. 

The introduction by Koch in 1881 of the plate method of sepa- 
rating bacteria paved the way for rapid advances in bacteriology, 
and+during the next ten years the bacterial causes of several 
diseases were discovered and proven by thorough test, and since 
then the number of diseases known to be due to bacteria has 
gradually increased. 

The history of immunity extends far back into ancient times. 
For many diseases it was recognized that those who recovered 
could associate with the sick without danger to themselves. 
Recognizing this, people sometimes exposed themselves purposely 
in order to have the disease at a convenient time. Artificial 
inoculation to cause small-pox was introduced into Europe from 
the Orient in 1721. The use of cowpox, vaccination, was discov- 
ered by Jenner in 1797. Artificial immunization by inoculation 
with altered bacterial cultures was first successfully demonstrated 
by Pasteur in chicken cholera and in anthrax in 1881. The 
practice of inoculation with dead bacterial cultures has’ become 
almost universal in the armies of the world since ror4, for the 
prevention of enteric fevers. Analogous methods have been 
devised for many other diseases. The discovery of the antitoxic 
property of the blood serum of animals immunized to tetanus 
and to diphtheria was made by von Behring and Kitasato (1891). 

With the discovery of amebz in the stools in tropical dysentery 
by Loesch (1875) and of the malarial plasmodium in the blood 
by Laveran (1880) the relationship of protozoa to important 
diseases was suggested. An enormous number of protozoal para- 


INTRODUCTION 13 


sites are now known, many of them associated with important 
diseases. The strict proof of causal relationship to the disease 
has presented greater difficulties here, especially the step of art fi- 
cial culture. However, the causal relationship of bacteria hav- 
ing been demonstrated, the probable causal relationship of 
the protozoa has found more ready acceptance. Cultures of 
ameba have been obtained by many workers but the successful 
cultivation of a pathogenic ameba is still questionable. Pure 
cultures of trypanosomes were obtained by Novy and his pupils 
(1903-04) and the infections again produced by inoculation with 
these cultures. 

The transmission of protozoal diseases by insects, first demon- 
strated by Salmon and Smith in Texas fever, has developed into 
a subject of prime importance. Malaria and the insect, Anophe- 
les, sleeping sickness and tsetse fly, Glossina, are important ex- 
amples of this relationship. 

Obermeier (1873) described a motile spiral organism in the 
blood of relapsing fever, the first known parasitic member of a 
group of very great importance. Very many pathogenic spiral 
organisms of this general type are now known. Their systematic 
relationships have not been fully worked out and further knowl- 
edge is necessary before they can be finally classed with either 
the bacteria or the protozoa. Artificial culture of these organisms 
has revealed a close relationship. to anaerobic bacteria, as far as 
nutritive requirements are concerned, the most successful culture 
methods being those devised by Noguchi (1912). Many of these 
parasites are transmitted by insects and they pass through a 
somewhat obscure development in the insect carriers, the forms 
developed being extremely minute (Nuttall, 1912). These facts 
suggest a possible relationship of this group of organisms to the 
filterable viruses. 

Nocard (1899) discovered that the virus of pleuro-pneumonia 
of cattle would pass through filters impervious to bacteria. The 
number of recognized filterable viruses has grown appreciably 


since then and among them are the causes of several very im- 
é 


14 INTRODUCTION 


portant diseases, such as yellow-fever, dengue fever, poliomy- 
elitis, measles, typhus fever, small-pox, rabies and hog cholera. 
Knowledge of this group of organisms is still relatively meagre 
and many features are still obscure or in controversy.. Micro- 
scopic methods of defining their form and structure are still poorly 
developed but they cannot with justice be regarded as wholly 
in the realm of the unknown. 

Agriculture——The importance of microbes in soil fertility 
and agriculture has a relatively short history. Duclaux, 1885, 
showed that plants could not well utilize complex organic matter 
as food in the absence of microbic life. In addition to ordinary 
decomposition of organic matter, bacteria also bear an important 
relation to the nitrogen metabolism of plants. Hellriegel and 
Wilfarth (1886-88) showed the infectious nature of the nitro- 
gen-fixing root tubercles of legumes, and the organism B. radicicola 
was isolated by Beyerinck in 1888. The importance for agricul- 
ture of other living elements in the soil, such as amebe and 
nematodes, has been more recently recognized. 

Although it is well to recognize the many important applica- 
tions of bacteriology, a word of caution may not be amiss, lest 
we follow too eagerly the alluring applications and neglect the 
secure foundation of scientific knowledge of the biology and bio- 
logical relationships of micro-organisms, the proper training in 
logical thinking concerning these beings and in the technic of 
dealing with them. 


PART | 
BACTERIOLOGICAL TECHNIC 


CHAPTER I 
THE MICROSCOPE AND MICROSCOPIC METHODS 


The development of bacteriology has depended specially: 
upon the development of new methods of scientific study, and 
in a very important way upon the improvements in construction 
of the microscope and in methods of preparing objects for study 
under the microscope. Knowledge of the construction of a micro- 
scope is not an essential part of bacteriology but the demands 
of modern microscopical methods require a skill in manipulation 
of the instrument which is best acquired after the principal struc- 
tural features of the microscope are understood. 

The Development of the Microscope.—Roger Bacon, in 
1276, seems to have been the first to recognize the peculiar prop- 
erties of a lens. Spectacles began to be used about the same 
time and are said to have been invented by d’Armato.! Galileo 
(1610) probably made the first record of the use of the com- 
pound microscope. It was a lens maker, Anton van Leeuwen- 
hoek, who first saw bacteria in 1683. A method of correcting 
chromatic aberration was discovered by Marzoli in 1811, but 
became generally known through the work of Chevalier in 1825. 
The correction of the color defects was accomplished by the com- 
bination of two kinds of glass, crown glass and flint glass, in the 
objective lens system, and made pos ible the construction of 
achromatic objectives, perhaps the most important advance ever 


1 Jour. A. M. A., Nov. 9, 1912, Vol. LIX, p. r72r. 
15 


16 BACTERIOLOGY 


made in the construction of the microscope. Abbé (about 1880) 
introduced his substage condenser which made possible the intense 
illumination of the microscopic field. In collaboration with 
Zeiss, Abbé (1886) devised an objective lens system with more 
perfect chromatic correction than had been previously attained. 
These objectives are constructed of several different kinds of 
glass and have in addition one lens composed of fluorite. They 
are called apochromatic objectives. Siedentopf and Zsigmondi 
(1903) devised a method of illuminating the microscopic prepa- 
‘ration by horizontal beams and so brought to view exceedingly 
minute refractive particles as luminous points on a dark field. 
The various dark-field condensers introduced in recent years 


Fic. 1.—The’formation of an image by means of a simple pin-point aperture. 
(After A. E. Wright.) 


(1906) utilize similar principles, the object being illuminated by 
oblique light. Recently, Gordon has devised the tandem micro- 
scope, an instrument which has demonstrated the possibility of 
achieving greater microscopic resolution than has previously 
been attained and even suggests that there is no necessarily 
final limit to the degree of magnification at which satisfactory 
definition and resolution may be achieved. 

Principle of the Microscope.—The formation of an image by 
means of a simple pin-point aperture is illustrated in Fig. 1. It 
will be noted that the magnification achieved is the quotient of 
aperture-image distance divided by object-aperture distance; 


THE MICROSCOPE AND MICROSCOPIC METHODS 17 


also that the sharpness of outline of the image increases and the 
brilliancy diminishes as the size of the aperture is decreased. 

If the simple aperture be replaced by a convex lens and the 
object and the screen be set at the conjugate foci of the lens, it 
will be seen that magnification is again the quotient of the aper- 


Fic. 2.— Image formation by a single lens, Note that the image, at the right 
is 36 the size of the object, in proportion to their respective distances from the lens; , 
the opening angle being 3 the size of the closing angle. 


ture-image distance divided by the object-aperture distance. 
The sharpness of outline, however, depends now upon the quality 
of the lens and the accurate adjustment of the distance, and 
brilliancy is not seriously impaired in attaining definition. 


Fic. 3.—Image formation by two lenses in series without magnification. Note 
that the opening angle of the beam proceeding from the object, at the left, is equal to 
the closing angle of the beam forming the image at the right. 


Image formation in the human eye is an example of the work- 
ing of the lens-armed aperture. The rays of light are brought 
to a focus on the retina and the image produced here is inverted 
and actually much smaller than the object, the reduction (minifica- 
tion) being again measured by the quotient of the lens retina 
distance divided by the object-lens distance. The longer the 


antero-posterior diameter of the eye, the larger will be the retinal 
2 


18 e BACTERIOLOGY 


image. Our subjective interpretation of the stimulation of the 
retina (i.e., what we see) is influenced by other psychological 
elements and especially by the memory of things seen before. 

When two lenses are disposed in series so that the rays of 
light coming from a point in the object pass through both lenses 


Fic. 4.—Image formation by two’ lenses in series, with magnification of two 
diameters. Note that the opening angle of the beam is twice as large as the closing 
angle. 


before coming to a focus, we find the possibilities shown in Figs. 3, 
4 and 5. In the figures it will be seen that the image produced 
when the first lens is in position so as to render the rays parallel 
(Fig. 4), is just five times as large as that produced when it is 


= 
— - 
aS 
— 
é ae 
Sra 
v4 aise = 
4 A SS RS 
—_ — = 
eS ce ae CEOs = 
<= _-_ wae = 
— — — 
a, = =a 
= SS se _~— SS 
SS — eo Ss 
SAX, Oe as 
a pcr a 


Fic. 5.—Image formation by two lenses in series, with magnification of three 
diameters. Note that the opening angle of the beam is three times as large as 
the closing angle. 


left out (Fig. 2), assuming that the second lens is capable of change 
so as to focus upon the same screen slightly divergent rays pro- 
ceeding from the object. It will further be perceived that the 
sine of the angle of divergence of the beam proceeding from the 
object varies directly with the magnification achieved, and further 


THE MICROSCOPE AND MICROSCOPIC METHODS 19 


that the magnification in any such system is equal to the quotient 
of the sine’ of the angle of divergence of the beam proceeding from 
the object, divided by the sine of the angle of convergence of the 
beam to form the image. This is capable of mathematical proof 
and is illustrated in the four figures. From these it is evident that 
magnification is a function of the relation of these two angles of 
the opening and closing limbs of the beam, and that the inter- 
mediate course of the rays, whether parallel, convergent or 
divergent, is negligible in this computation. If the second lens 
be that of the eye and an image is to be formed on the retina, then 
the rays proceeding from a point must be rendered parallel, ‘or 
approximately so, by the first lens. This is the arrangement 
which exists in the simple microscope or in the ordinary reading 
glass. The magnification achieved by such a simple microscope 
is measured by the relation between the magnitude of the ‘mage 
on the retina when the lens is employed, and the size of such an 
image when the lens is left out of the path of the light. The 
value of the reading glass, entirely aside from considerations of 
magnification, in conditions of hyperopia and presbyopia is also 
evident from these figures, as it of course renders the rays coming 
from a near point more nearly parallel, and thus enables the re- 
fracting media of the presbyopic eye to bring them to a focus. 

So far we have been employing in our discussion the ideal lens, 
one which refracts all light equally and brings to a focus in one 
plane all rays proceeding from one plarie in the object. As 
a matter of fact the ideal lens in this sense does not exist. The 
simple convex lens has many serious optical defects. 

Points in the same plane in the object are imaged by the simple 
lens on a curved surface, the segment of a spherical surface. 
This defect is known as spherical aberration. It is diminished 
to some extent by combining convex and concave lenses and the 


1In the figures, as drawn, this statement actually applies to the tangents of the 
angles designated, rather than the sings. However, for very small angles the sine 
and tangent are approximately equal. The use of the term sine finds its complete 
justification in the fact that the plane at which the rays are bent is not flat but is 
the segment of a sphere or its optical equivalent. 


20 BACTERIOLOGY 


correction may be changed by altering the distance between these 
component lenses, as, for example, in an objective equipped with 
a correction collar. Objectives corrected in respect to spherical 
aberration are designated as aplanatic. Restriction of the size 
of the field is also an important factor in making it appear flat. 

Light of different wave lengths (different colors) is refracted 
to a different degree by the simple lens, so that, for example, the 
violet rays are brought to a focus earlier than the red rays, with 
the remainder of the spectrum spread out between. This defect 
is known as chromatic aberration. It is corrected to a very con- 
siderable extent by combining biconvex lenses of crown glass 


Fic. 6.—Microscope objectives showing the component parts of the objective lens 
system. 


with plano-concave lenses of flint glass (achromatic objectives), . 
to a still nicer degree by combinations of lenses of several different 
kinds of glass together with a lens of fluorite (apochromatic ob- 
jectives); and finally, when desired, chromatic aberration may 
be wholly avoided by employing mono-chromatic light. 

A third defect of lenses is known as diffraction, which is a 
phenomenon giving rise to a whole group of less luminous second- 
ary images around the principal image. The influence of diffrac- 
tion is most evident when the surfaces of the lens are roughened 
by scratches or by presence of dust, but even the most perfect 
lens systems are not wholly free from diffraction phenomena. 


THE MICROSCOPE AND MICROSCOPIC METHODS 21 


SS 
NY 


i 
Hl 
i 
HI 


iy 


Fic. 7.—Sectional view of a compound microscope illustrating the course of 
two beams proceeding from two points in the object (P and Q) and indicating the 


subjective interpretation of the image formed on the retina. : 


22 BACTERIOLOGY 


Some of these defects will require brief consideration in our 
discussion of the compound microscope. 

In the modern compound microscope the beam of light pro- 
ceeding from a point in the object is refracted by the lens system 
of the objective (Fig. 6) so as to render the rays slightly conver- 
gent. Near the upper end of the tube of the microscope these 
rays are further refracted by the lower lens of the eye-piece and are 


Fic. 8.—Image formation in the compound microscope. Compare with Fig. 9. 


converged and brought to a focus in the interior of the eye-piece. 
A screen placed at this level would show a real image, and any 
pattern (for example an eye-piece micrometer) inserted in the 
eye-piece at this level is readily fused with the microscopic field. 
Continuing in a straight line the rays diverge from this focus to 
reach the upper lens of the eye-piece. In traversing this lens 


Fic. 9.—Image formation in the compound microscope with an eye-piece of 
higher power. Observe that the increased magnification is accomplished by narrow- 
ing the beam of light which enters the eye and so diminishing the size of the closing 
angle. Compare with Fig. 8. 


‘they are again refracted and made parallel so that they will 
enter the eye and be brought to a focus on the retina. The paths 
of two beams of light, one proceeding from the center of the micro- 
scopic field and one from its periphery, are illustrated in Fig. 
8. Fig. 9 shows the change which is introduced by the use of 
an eye-piece of higher magnifying power. 

It will be noted that the objective and lower lens of the eye- 


THE MICROSCOPE AND MICROSCOPIC METHODS 23 


piece bring the beam to a focus forming a real image, and that 
the rays diverging again from this image are again brought to a 
focus on the retina by the upper lens of the eye-piece and the 
optical structures of the eye. The magnification represented in 
the first image is the quotient of the sine of the angle of the opening 
limb of the beam divided by: the sine of the closing angle. The 
subsequent magnification between this and the eye is the quotient 
of the sine of the opening angle of the rays proceeding from this 
image divided by the sine of the closing angle of the rays approach- 
ing the retina. The closing angle at the formation of the first 
image and the opening angle of the beam proceeding from it are 
obviously equal, so that the total magnification equals the sine 
of the first opening angle divided by the sine of the last closing 
angle in the system. It will be noted that the eye-piece of higher 
power narrows the beam and decreases the closing angle. 

By placing the eye about two feet above the eye-piece, one 
may measure the width of the beam as it emerges from the latter. 
Now by changing to an eye-piece of higher power he can readily 
demonstrate the consequent narrowing of the beam. This 
narrowing of the beam makes more important the obscuration 
caused. by any defect in the optical system of the eye itself. 
Microscopic vision is disturbed by floating shadows and hazy 
definition due to such partial obscuration especially when the 
beam has been too much narrowed. For ordinary work, therefore, 
an eye-piece of low power will prove more satisfactory. 

In the above discussion, the refractive index of the vitreous 
humor has been disregarded. This is not the same as that of 
air (in reality it is about 1.3) and the peripheral beam is there- 
fore bent toward the axis of the eye instead of proceeding in its 
former direction, the magnification being thereby reduced by 
precisely the fraction : 


refractive index of air got 


refractive index of vitreous 1.3 


This brings us to a definition of numerical aperture. The numer- 


‘ 


24 BACTERIOLOGY 


ical aperture of the closing limb (n.a.) is the sine of half the angle 
of the converging beam multiplied by the refractive index of the 
medium (in this instance the vitreous humor). This is commonly 
designated as n.a. The numerical aperture of the opening limb 
of the beam (N.A.), proceeding from a point in the object to the 
objective, is the sine of half the angle of this beam multiplied by 


Fic. 10.—Central illu- Fig. 11.—Illumi-: Fic. 12.—Ilumination by 
mination by a narrow nation by a hollow abroad beam converging upon 
beam. Three beams of cone of light converg- the object at a wide angle. 
parallel rays, such as might ing upon the object Only a few beams of parallel 
come from a large white at a wide angle, by rays from a distant point 
cloud, are represented. use of the centralspot source of light are represented 
Note that these raysreach stop. Compare with inthe figure. Compare with 
the object as almost verti- Fig. 14, and with Fig. Fig. 17. 
cal rays, varying from the 16. 
vertical by only a narrow 
angle. Compare with Fig. 

IS. 


the refractive index of the medium through which it passes. 
This is commonly designated as N.A. Many desirable properties 
of objectives, other than magnification, such as brilliancy of 
illumination, definition, and resolution in depth, also depend upon 
the numerical aperture, which is therefore :perhaps the most 
important single feature of objectives of high power. 

Another important optical part of the bacteriological micro- 


THE MICROSCOPE AND MICROSCOPIC METHODS 25 


scope is the substage illuminating apparatus, consisting of the 
mirror, the iris diaphragm and the condenser. These are neces- 
sary to illuminate minute objects so that they may be satis- 
factorily studied at high magnifications. By the use of the iris 
diaphragm and of the central spot stop, the ordinary condenser 
may be made to furnish three different kinds of illumination, (1) 
central illumination by a narrow beam, (2) illumination by a 
hollow come of light converging on the object at a wide angle, an 
example of dark-field illumination, and 
(3) intense illumination by a broad 
beam converging at a wide angle upon 
the object. These . possibilities are~ 
illustrated in Figs. 10, 1: and 12. 
Dark-field illumination is obtained in 


up oo 
Fic. 14.—Optical parts of 
the dark-field condenser with 
object slide and microscope ob- 
: jective with funnel stop in possi- 
Fic. 13.—Dark-field condenser showing tion. The path of light rays 
optical parts and centering mechanism. is indicated by the dotted lines. 


a more satisfactory manner by employing a special condenser 
made for the purpose, illustrated in Figs. 13 and 14. The way 
in which these different methods of illumination affect the visi- 
bility of a colorless refractive object is illustrated in Figs. 15, 16 
and 17. 

Visibility of Microscopic Objects.—In the use of the micro- 
scope it is necessary to pay some attention to the factors upon 
which visibility depends. An object may be distinguished and 
perceived by the eye only when the light coming from the object 


26 BACTERIOLOGY 


differs from that coming from its surroundings either in quantity 
or in quality, and the greater the extent of this difference the 
more distinctly visible will the object be. Uncolored trans- 
parent objects are visible by virtue of their ability to refract light 
and so to present darker and lighter zones. If the surrounding 
medium possess the same refractive power as the colorless trans- 


Fic. 15.—Showing the manner in which the ‘‘dark outline picture"’ is produced. 
(After A, E. Wright.) 


parent object, the latter is invisible.!_ Microscopic objects may 
conceivably be invisible or so nearly invisible as to have escaped 
detection for this very reason. If, however, the object be 
suspended in a medium of lower refractive index, then it may be 
defined by light and shade, and it is most clearly defined when 
illuminated in one of two ways, either by a rather narrow direct 


+ This may be illustrated fairly well by immersing clean, perfectly clear glass 
beads in oil of cedar wood. 


THE MICROSCOPE AND MICROSCOPIC METHODS 


Fig. 16.—Showing the manner in which the “‘bright outline picture’’ is produced. 
(After A. E. Wright.) 


Fic. 17.—-Showing the manner in which the outlines are obliterated when an object 
js illuminated by a homogeneous illuminating field. (After A. E. Wright.) 


28 BACTERIOLOGY 


beam of light passing from behind it directly toward the eye, in 
which case the object is defined by dark outlines upon a white field; | 
or by oblique beams directed at an angle from the sides, when the 
object is defined by bright outlines on a dark background. Ii, 
however, the object be illuminated from all sides or from behind and 
from both sides by light of similar intensity, its outlines become 
less distinct and may even be completely obliterated so that the 
object becomes invisible. These facts may be crudely illustrated 
by holding a test-tube full of water, (1) between the eye and a 
window, (2) between the eye and a dark wall between two win- 
dows, and (3) against the center of the window pane. Their 
importance in microscopy may be readily illustrated by examining 
a simple preparation of living bacteria, (1) with the iris diaphragm 
nearly closed, (2) with the dark-field condenser, and (3) with the 
ordinary condenser with the iris wide open. It will be evident 
that the third arrangement is fatal to the definition of colorless 
transparent microscopic objects. It will also be observed that 
the dark field offers an advantage in the ease with which the ob- 
jects can be seen, the small luminous outline on the dark back- 
ground being more distinct than the dark outline on the luminous 
background. The former might be compared in this respect 
to a star at night, and the latter to a sun spot in the daytime, 
which though many times larger may not be readily perceived. 

The method of making objects visible by a difference in quality 
of light (color) usually involves the necessity of staining. Colored 
preparations have certain very important advantages for micro- 
scopic study. If an object can be differentially colored, that is, 
stained a different color or a different shade. of the same color 
from the material by which it is surrounded, it becomes clearly 
visible even in the absence of different refractive power. Refrac- 
tion may be largely eliminated by replacing the fluids of the 
preparation by other fluids of high refractive index, such as 
cedar oil or balsam, and this elimination of refraction eliminates 
the opacity of the preparation, “clears” it, and makes possible the 
distinct definition of minute objects situated in the deeper optical 


THE MICROSCOPE AND MICROSCOPIC METHODS 29 


planes of the preparation. A proper appreciation of this micro- 
scopical principle will at once suggest the importance of differential 
staining methods in microscopy. 


Fic. 18.—Microscope. 


The Bacteriological Microscope.—The bacteriological micro- 
scope consists of a tubular body which carries the optical parts, and 


1 This principle may be crudely but clearly illustrated by covering a ‘colored 
glass bead with a deep layer of colorless glass beads in a test tube, so as to conceal 
the former when looked at from above. By replacing the air between the beads 
with homogeneous cedar oil the layer of colorless beads is rendered perfectly trans- 
parent and the colored bead is distinctly seen from above. Filter paper may be 
rendered transparent in a similar way. 


30 BACTERIOLOGY 


which can be raised or lowered for focusing. The objectives 
should be three in number, and should be attached to the body by 
means of a triple nose-piece, which permits any objective to be 
turned into the optical axis at will. The eye-piece slips into the 
upper and opposite end of the body or tube. The arrangements 
for focusing consist of a rack and pinion, which accomplish the 
coarse adjustment, and a more delicate fine adjustment. The 
stage, upon which the objects to be examined are placed, has an 
dpening in the middle. In this opening an iris diaphragm and 
Abbé condenser are inserted. The iris diaphragm enables one 
to alter the size of the opening as desired. Beneath the stage is a 
movable mirror, of which one side is plane and the other concave. 
All of these parts are supported on a short, heavy pillar which is 
fixed in the horseshoe-shaped base. 

The essential parts of the microscope are, of course, the eye- 
piece (German, Ocular), and the objective. Objectives are given 
various names by different 
makers, for instance, A, B, C, 
etc., or 1, 2, 3, etc.; or they are 
named according to their focal 
distances, as 24 inch, 14 inch, 14 
inch, etc. In_ bacteriological 
_ Fic. 19.—Abbé Condenser. On the work a rather “low power” 24 
ss side the figure gives a sectional ai 34 inch objective, een dinary 

“high power” lé to 4 inch dry 
objective, and a high power 1%» inch oil-immersion objective are 
needed. The magnification with the 24 or 34 inch objective is 
about 75 to 1oo diameters; with the 1 to 1¢ inch 400 to 700 
diameters; with the }{2 immersion 750 to 1,000 diameters. The 
magnification varies according to the eye-piece used, as well as 
with the objective. A 1 inch and 114 inch eye-piece (Leitz No. 
2 and No. 4) serve well for most purposes. The eye-pieces are 
usually named arbitrarily, like thé objectives. The oil-im- 
mersion objective is used in the examination of bacteria where a 
very high power is desired. A layer of thickened oil of cedar- 


THE MICROSCOPE AND MICROSCOPIC METHODS 31 


wood is placed between the lower surface of the objective and 
the upper surface of the glass covering the object under ex- 
amination. The oil must be wiped away from the surface of 
the objective when the examination is finished. For this purpose 
the soft paper sold by dealers in microscopical apparatus serves 
admirably. Care must be taken not to scratch the lower surface 
of this objective. Oil of cedar-wood furnishes a medium having 
nearly the same refractive index as the glass of the lens and the 
glass on which the object is mounted, and it obviates the dispersion 
of light which takes place when a layer of air is interposed between 
the objective and the object, as happens with the ordinary dry 
lens. 

The microscope should be placed in front of the observer on 
a firm table. The observer should be able to bring the eye easily 
over the eye-piece when the tube of the microscope is in vertical 
position. Daylight should be employed if possible. When arti- 
ficial illumination is necessary, an ordinary lamp, a Welsbach 
burner or an incandescent electric light may be used. It is well 
to modify the artificial light by inserting a sheet of blue glass be- 
tween the light and the mirror or to employ a special incandescent 
nitrogen bulb made of ‘‘daylight” glass, which can be obtained 
from dealers in electric lamps. . 

In order to focus upon any object, having first secured a satis- 
factory illumination with the mirror, it is best, beginning with 
the low power and using the coarse adjustment for focusing, to 
bring the objective quite close to the object, and then, with the 
eye in position, to raise the tube until the object comes into focus. 
The exact focusing is done with the fine adjustment. The -ob- 
server should keep both eyes open when using the microscope, 
and should be able to use either eye at will. 

All measurements of microscopic objects are expressed in 
terms of a micromillimeter. This is one-thousandth of a milli- 
meter (0.001 mm.), which is about }45900 of an inch. This unit 
is designated as a micron, and is denoted by the Greek letter u. 
For example, 54 = 0.005 mm. = 14 9 90 inch. 


32 BACTERIOLOGY 


The Platinum Wire.—The substance under examination. — 


is placed upon thin slips of glass called cover-glasses or directly 
upon thicker strips of glass, called slides. The material is spread 
over the glass by means of a platinum wire which has been fixed 
in a glass rod about six inches long. Such a platinum wire is used 
constantly in doing bacteriological work. The 
°) platinum wire must be stiff enough not to bend too 
easily, and yet it should not be so large that it 
will not cool rapidly after heating. A good size 
for most purposes is No. 28, English standard 
gauge, diameter .or4 inch. Instead of the expen- 
sive platinum wire, one may use nichrome wire, 
which is much cheaper and serves as well for all 
ordinary purposes. The wire may be straight 
throughout its length, or the tip may be bent to 
form a loop. It is well to follow, from the begin- 
ning, certain rules which make the use of the wire 
safe and accurate. Every time it is taken into the 
hand and before using it for any manipulation, 
heat it in the flame of a Bunsen burner or an 
alcohol lamp to a red heat; and always, after 
using and before putting it down, heat it again to 
ta ved heat. After the needle has become wet by 
Fic. 20.—Need- dipping it in a fluid and is to be sterilized in the 
les used for inocu- rae : 
lating media. flame, it is necessary to avoid “‘sputtering” of the 
fluid by bringing the wet needle gradually to the 
flame, so as to dry the material adhering to it before burning it 
or, better, by holding it within the central cooler cone of the 
Bunsen flame to accomplish the same purpose. This procedure 
must be done with great care when the wire has been dipped in 
milk or other substances containing oil. When the needle 
“‘sputters,”’ as it is called, from too rapid heating, particles that 
have not yet been sterilized may be thrown some distance. On 
no- account. should the needle touch any object other than that 
which it is intended it should touch. With such a platinum 


THE MICROSCOPE AND MICROSCOPIC METHODS 33 


wire, which has been properly sterilized, one can easily remove 
portions from a culture of bacteria, or from a fluid in which 
bacteria are supposed to be present. The rod in which the wire 
is fixed should be held between the thumb and forefinger of the 
right hand like a pen. . 

Glass Pipettes.—Sterile glass tubes drawn out to form slender 
capillaries, Pasteur pipettes, are very convenient instruments 
for handling bacteriological materials, and, for many kinds 
of work, really indispensable. They serve nearly all the pur- 
poses of the platinum wire and are capable further of use to 
transfer large quantities of fluid without contamination. They 
are also especially useful in collecting material from patients 


Fic. 21.—Drawn-out tube pipettes of Pasteur. a, Plugged, sterile tube aj 
kept in stock; b, the same heated at x in blast-lamp and drawn out; then sealed at 
x; c and d, completed pipettes; e, the same with bulb. (After Novy.) 


and at autopsy. Each pipette is sterilized and discarded after 
use.. : 

These pipettes are made by cutting glass tubing of a suitable 
size, diameter 3 mm. to 9 mm., into pieces from.zo to 40 cm. in 
length. The cut ends are smoothed in the flame. In the tubes 
of larger caliber it is well to make a constriction about 5 cm. from 
each end. Each end is plugged with cotton. The tubes are then 
sterilized by dry heat. By heating the middle of the tube in 


a blast lamp or over a large Bunsen flame, the glass may be 
3 


34 BACTERIOLOGY 


softened and then drawn out into a capillary of any desired 
length and caliber. This is melted in the middle and severed 
by the flame, giving two pipettes. When a large capacity is 
desired a bulb may be blown in the tube between the capillary 
and the cotton plug. This requires a little practice. The tip 
of the pipette is finally broken off with aid of a file, sterilized by 
the flame and the pipette is ready for use. The various steps in 
the preparation of pipettes are illustrated in the figures (Fig. 21). 

The Hanging-drop.—Living bacteria may be studied with 
the microscope while suspended in some fluid substance. The 
platinum loop having been heated to a red heat in the flame and 
having been allowed to cool, a small portion of the culture or 
other material may be removed with-it and deposited in the center 
of an ordinary cover-glass. The needle should again be sterilized” 
in the flame. When cultures on solid media are to be examined, 
a small particle may be mixed with a drop of sterilized water or 


TMT 


FiG, 22, 


bouillon. The cover-glass should have been carefully cleaned and 
sterilized over the flame. The cover-glass with the small drop 
of fluid material held in sterilized forceps is now to be inverted 
over a sterilized glass slide, which has a concavity ground in the 
middle of it. Around the concavity, the slide should be smeared 
with vaseline. In this manner a small air-tight chamber is made. 
This slide and cover-glass is next put upon the stage of the micro- 
scope. A good dry lens, if of sufficiently high power, is more 
convenient for examining the hanging-drop than an oil-immer- 
sion. If the latter be used, having placed a drop of cedar-oil 
on the center of the cover-glass, and a good light having been 
secured, the oil immersion objective should be brought down 
upon this drop of oil. The beginner often experiences difficulty 
in focusing upon a hanging-drop. It is necessary to shut off 
most of the light by means of the iris diaphragm, for as has 


THE MICROSCOPE AND MICROSCOPIC METHODS 35 


already been pointed out (page 26), colorless objects may be clearly 
seen only when illuminated either by a narrow central beam or 
by oblique illumination (dark-field). Often it is well to secure 
the focus roughly upon the extreme outer edge of the chamber, 
or to find the edge of the drop of fluid with the low power and 
then_focus upon this edge with the oil-immersion objective. 
Above all things guard against breaking the cover-glass by forcing 
the objective down upon it. The motility of certain bacteria is 
one of the most striking phenomena to be observed in the hanging- 
drop. It is not to be confused with the so-called “Brownian 
movement” which is exhibited by fine particles suspended in a 
watery fluid. It is well for the beginner to observe the character 
of the Brownian movement by rubbing up some carmine in a 
little water, and with the microscope to study the trembling 
motion exhibited by these particles of carmine. It will be noticed 
that, although the particles oscillate, no progress in any direction 
is accomplished unless there are currents in the fluid. Such cur- 
rents might give rise to the impression that certain bacteria 
possessed motility when they were, in fact, powerless to move 
of themselves. In the hanging-drop the multiplication of bacteria 
can be studied, the formation of spores and the development of 
spores into fully formed bacteria. The hanging-drop is also 
used extensively for the demonstration of the agglutination 
reaction with the bacillus of typhoid fever. Sometimes bacteria 
must be watched in the hanging-drop for hours, or even days, 
and it may be necessary to keep it at the temperature of the human 
body for this length of time. Various complicated kinds of 
apparatus have been devised for this purpose, but they are needful 
only for special kinds of work. When the hanging-drop prepara- 
tion is no longer required, the slide and cover-glass should be 
dropped into a 5 per cent carbolic acid solution and afterward 
sterilized by steam. 

The Hanging-block.—Hanging-block preparations, which 
were introduced by Hill,! make use of a cube of nutrient agar 

1 Journal of Medical Research, Vol. VII, March, 1902. 


36° BACTERIOLOGY 


instead of a drop of fluid. Bacteria are distributed on the sur- 
face of the agar, which is then applied to a cover-glass, and 
mounted like a hanging-drop. The bacteria are thus kept in a 
layer close to the glass, where growth may be studied. 

The Microscopic Preparation for Study by Dark-field Illumi- 
nation.—The central portion of a clean glass slide is encircled 
with a ring of vaseline, and a drop of the fluid to be examined’ 
is deposited on the clean surface in the center of the ring by means 
of a capillary tube. It is then covered with a clean large cover- 
glass so that the fluid spreads out in a moderately thin layer 
beneath the cover-glass and is confined on all sides by the vaseline, 
thus preventing evaporation and resulting currents in the 
preparation. 

Best results with the dark-field microscope are obtained only 
in a dark or dimly lighted room. An electric arc or a powerful 
.gas-light or, better still, a powerful daylight-glass nitrogen-bulb 
incandescent electric light, may be employed as the source of light, 
and it is well to put a flask of water between the light and the 
microscope to eliminate the heat-rays. The substage condenser 
of the microscope is replaced with the special dark-field condenser 
and this is carefully centered. A large drop of immersion oil is 
placed on the upper surface of the condenser. The slide is 
carefully placed upon the stage so that the oil fills in completely the 
space beween the condenser and slide and remains free from air- 
bubbles. The preparation is then ready for examination. Objec- 
tives of numerical aperture wider than 1.0 cannot be successfully 
used with the ordinary dark-ground condensers and therefore it is 
necessary to stop down the aperture of the oil-immersion objec- 
tive before using it. A special funnel stop is furnished for this 
purpose. When this has been attached the preparation may be 
studied with the oil-immersion objective in the usual way. Skill 
in this method of studying unstained microbes is quickly acquired, 
offering, as a rule, less difficulty than the method of central 
illumination which is employed for the hanging-drop and hanging- 
block. 


THE MICROSCOPE AND MICROSCOPIC METHODS 37 


Smear Preparations for Staining—The examination of 
bacteria with the microscope is carried out to a very large extent 
by means of smears made upon thin slips of glass. Such slips 
of glass are generally called cover-glasses. It is best to obtain the 
kind sold by dealers as No. 1, 34 inch squares. 

The cover-glass may be cleaned best by immersion in a mix- 
ture of sulphuric acid and bichromate of potassium solution, and 
afterward washed thoroughly in distilled water, and finally in 
alcohol. A stock of clean cover-glasses may be kept in a bottle 
of alcohol, or perhaps preferably in alcohol containing 3 per cent 
of hydrochloric acid. 


CLEANING FLuIp 


Potassium bichromate.................... 40 grams, 
Water ovina sien adabiies weenadetone Caan res 1§0 C.c. 
Dissolve the bichromate of potassium in the 

water, with heat; allow it to cool; then add 

slowly and with care sulphuric acid, com- 

METClAls ch Guiauinactardind hatasse scene ot 230 C.C. 


When they are needed for use they should be wiped clean 
with a piece of linen cloth. As a rule, cover-glasses cleaned in 
this way still retain a small amount of oily matter on their surfaces, 
sufficient to prevent the proper spreading of a drop of water. 
This difficulty may be overcome by passing each glass several 
times through the flame. It is better, when time permits, to fill 
an Esmarch dish with clean cover-glasses and then heat them in 
the oven at 200°C. for half anjhour. Cover-glasses treated in this 
way will allow the droplet of bacterial suspension or other material 
to spread perfectly. They must be carefully preserved in a 
covered dish from which they are to be removed only by clean 
(flamed) forceps. Carelessness in this matter may necessitate 
recleaning of the whole lot of cover-glasses. 

An ordinary pair of fine forceps may be used to pick up the 
cover-glass and insert it between the blades of such special forceps 
as those of Cornet or of Stewart. Perhaps the most convenient 
style of forceps is that devised by Novy, provided with a clasp. 


38 BACTERIOLOGY 


Bacteria may be placed upon the cover-glass by allowing the 
glass to fall upon one of the colonies of bacteria, on a gelatin or 
agar plate (see page 110), which will adhere to it in part, produc- 
ing an “impression preparation” (German, Klatschpreparat). 
Such a preparation, after drying in the air, is to be fixed by pass- 


Fic. 23.—Cornet forceps for cover-glasses. 


ing it through the flame three times. (See below). The forceps 
with which it is handled should be sterilized in the flame. 
Generally bacteria contained in fluids, like sputum, or taken 
from the surface of a culture, are smeared over the cover-glass 
by means of the platinum wire or loop, which must be heated to 


Fic. 24.—Stewart forceps for cover-glass. 


a red heat before and after the operation. Such preparations 
are called smear, cover-glass, cover-slip, or film preparations. 
When the material to be spread is thick or very viscid, a small 
drop of distilled water must first be placed in the center of the 
cover-glass so as to dilute it. Begitiners generally take too much 


Fic. 25.—Novy’s cover-glass forceps with clasp. (Afler Novy.) 


material on the wire. As thin a smear as possible is made. It 
is allowed to dry in the air; this should occupy a few seconds. 
The drying may be hastened by holding the forceps with the 
cover-glass a long distance above the flame, at a point where the 


THE MICROSCOPE AND MICROSCOPIC METHODS 39 


heat would cause no discomfort to the hand. Having dried 
the preparation, it is to be passed through the flame of a Bunsen 
burner or alcohol lamp three times, taking about one second for 
each transit. The heat of the flame serves to dry the bacteria 
upon the cover-glass and fix them permanently in position; it is 
not sufficient, however, when applied in this manner, to kill all 
kinds of bacteria, especially those containing spores. After it 
has been passed through the flame three times the preparation 
may be stained with one of the aniline dyes, and after washing 
in water and drying, may be mounted, face down, in Canada 
balsam upon a glass slide. It makes a suitable object to be ex- 
amined with the oil-immersion objective. The slide is a thin 
slip of glass, 3 inches by 1 inch, with ground edges. 

The smear preparation may equally well be made directly 
upon the glass slide provided this be cleaned and heated to insure 


Fic. 26.—Kirkbride forceps ‘for holding slides. 


a clean surface free from oily matter. The fixation in the flame 
must then occupy a longer time than with the small and thin 
cover-glass. Such preparations have the advantage that several 
may be made upon one slide, and that after staining them they 
may be examined in cedar-oil, with the oil-immersion lens, without 
the use of the cover-glass and Canada balsam. They are also 
less readily broken in handling. Special care should be exercised 
_ to avoid soiling the surface of the objective with the material on 
the slide, for inattention to this matter may permit the confusion 
of material from two different specimens, a bit being carried from 
one smear to another by the lens. The forceps of Kirkbride will 
be found convenient when staining on the slide. The aluminium 


40 BACTERIOLOGY 


dish devised by Krauss,! or some similar dish, will be found useful 
when the stain has to be heated. Experiments have shown that 
the ordinary method of fixation in the flame, when applied to 
bacteria spread upon slides, has little effect on the vitality of many 
species. The beginner is, therefore, advised to make his prepara- 
tions on cover-glasses. 

When very resistant or dangerous pathogenic bacteria are 
being handled, after fixation by heat upon the slide or cover- 
glass, the preparation may, if desired, be immersed in 1—1000 
solution of bichloride of mercury long enough to kill the bacteria, 
without injuring the preparation or its staining properties. 

Staining Solutions.—The staining of bacteria is done for the 
most part with the aniline dyes. The object of staining bacteria 
is to give them artificially some color which makes them distinct 
and easily visible without imparting this color to the substance 
or medium in which they are imbedded. The substances known 
as aniline dyes are derivatives of coal-tar, but not always of aniline. 
These dyes are of great importance in bacteriological “ work. 
Their number is very large, but only a few are in common use. 

It is simplest to classify the aniline dyes as acid or basic. 
Eosin, picric acid and acid fuchsin are acid dyes; they tend to stain 
tissues diffusely. Fuchsin, gentian-violet and methylene blue are 
basic dyes; they have an affinity for the nuclei of tissues and for 
bacteria; they therefore are the dyes used chiefly in bacteriological 
work. The other varieties may be employed as contrast-stains; 
another contrast-stain frequently used is Bismarck brown. 
It is best to keep on hand saturated solutions of the aniline dyes 
in alcohol, which are permanent, but cannot be employed directly. 
for staining. In order to prepare the simple staining solutions, 
the alcoholic solution is diluted about ten times, or so as to make 
a liquid which is just transparent in a layer about 12 mm. in 
thickness, after filtering. [These watery solutions deteriorate | 
after a few weeks. 

Fuchsin and gentian-violet stain rapidly and intensely. 

1 Krauss, Jour. A. M. A., Apr. 6, 1912, Vol. LVIII, p. 1013. 


THE MICROSCOPE AND MICROSCOPIC METHODS 41 


Methylene blue works more slowly and feebly; it is to be preferred 
where the bacteria occur in thick or viscid substances, like pus, 
mucus, and milk. 

Aniline-water Staining Solutions—The intensity with which 
aniline dyes operate may be increased by adding aniline oil to 
the solution: 


Mix, shake vigorously, filter through wet filter paper. The fluid 
after filtration should be perfectly clear. Add— 


Alcoholic solution of fuchsin (or gentian violet, or 
methylene: blue) sii veces de ness fae bee aden ICC. 
Aniline-water staining solutions do not ‘keep well, and need to 
be freshly prepared about every two weeks. The applications 
of the aniline-water stains will be given under separate headings. 
In general, however, they are employed where a stain of unusual 
power is required. 

Sterling’s Gentian Violet.— Mix 2 c.c. aniline oil with 10 c.c. of 
‘95 per cent alcohol and add 88 c.c. of distilled water. Grind 
5 grams of gentian violet in a mortar, slowly adding the mixed 
liquid while grinding. Filter. The solution keeps and stains 
rapidly in Gram’s method. 

Carbol-gentian-violet—Grind 1 gram gentian-violet in a 
mortar with ro c.c. absolute alcohol; then add 2 grams pure 
crystals of carbolic acid and mix. Add distilled water, 70 c.c., 
and transfer the whole to a clean bottle. Rinse the mortar with 
a further 30 c.c. of distilled water and add this to the contents of 
the bottle. Filter into a clean bottle after 24 hours. This solu- 
tion keeps well and is used for staining by Gram’s method. 

Carbol-fuchsin.—The intensity of staining may also be in- 
creased by the presence of carbolic acid. The most common 
example of this is carbol-fuchsin. 


Saturated alcoholic solution of fuchsin............ Io C.c. 
5 per cent aqueous solution carbolic acid.......... 100 C.C. 


* 


’ + 
42 BACTERIOLOGY 


This solution keeps for some months. It is employed especially 
where very intense action is required, as in staining spores, flagella, 
and acid-proof bacteria. 

Loffler’s Methylene Blue.—A very useful solution, which 
keeps well, is Loffler’s alkaline methylene blue: 


Saturated alcoholic solution of methylene blue..... 30 C.C. 
T-10,000 aqueous solution of potassium hydroxide.. too c.c. 


This solution stains more intensely than simple methylene blue, 
and also gives rise to useful differential staining in smears and 
even in sections of tissue. 

Nocht-Romanowsky Stain.—This requires two solutions, one 
of ripened alkaline methylene blue, the other of eosin. 


Solution 1. 


Methylene blue.............. 0.00.00 0c eee I.0 gram. 
Sodium carbonate..............02 002 see 0.5 gram. 
Distilled Water ss scone yaadmnrace ioe gume diace sean nese 100.0 grams. 


Heat at 60° C. for two days until solution shows a slight purplish 
color. 
Solution 2. 


Eosin, yellowish, water soluble.............. 1.0 gram. 
Distilled Water's comune vaneevecmesyeeee es 100.0 C.C. 


In staining, a few drops of each of these solutions are mixed with 
about to c.c. of distilled water in an Esmarch dish, and the smear, 
which has previously been fixed in absolute methyl alcohol, is 
floated on this mixture for about ten minutes. Considerable 
practice is necessary before the best results are obtainable. 
The method is especially useful in staining blood films, and 
protozoa in blood, in feces or in culture. 

Leishman’s Stain.—Leishman has utilized the principle of 
Jenner’s stain! and has added to it the important additional 
constituents found in polychrome methylene blue by substituting 
this for the ordinary methylene blue used by Jenner. 


. *Jenner (Lancet, 1899, I, p. 370) first employed the solution of eosin and methy- 
lene blue in methyl] alcohol as a stain for blood films. 


THE MICROSCOPE AND MICROSCOPIC METHODS 43 


Solution A.—To a 1 per cent solution of medicinally pure 
methylene blue in distilled water add 0.5 per cent sodium car- 
bonate and heat at 65° C. for 12 hours, then allow it to stand 10 
days at room temperature. 

Solution B.—Eosin extra B. A. (Griibler) 0.1 per cent solution 
in distilled water. 

Mix Solutions A and B in equal amounts and allow to stand 
six to twelve hours, stirring at intervals. Filter and wash the 
precipitate thoroughly. Collect, dry and powder it. 0.15 gram 
is dissolved in 100 c.c. of pure methyl alcohol to form the staining 
solution. It keeps perfectly for at least five months. To stain, 
cover the dried but unfixed film of blood with the staining solu- 
tion. After 30 to 60 seconds add about an equal amount of 
distilled water. Allow this mixture to act for five minutes. 
Wash in distilled water for about one minute, examining the 
specimen mounted in water under thé microscope. Blot, dry 
thoroughly, mount in balsam, or preserve the specimen as an 
unmounted film. 

Numerous imitations or modifications of Leishman’s stain 
have been described. 

Giemsa’s Stain.—This stain contains certain of the essential 
constituents of polychrome methylene blue and eosin, the whole 
being dissolved in a mixture of glycerin and methyl alcohol. 
Giemsa’s Azur I is the substance methylene azure and his Azur 
II is this substance mixed with an‘equal amount of methylene 
blue. His Azur II-eosin is the compound precipitated when 
aqueous solutions of Azur II and eosin are mixed. The Giemsa 
solution is made according to the following formula: 


AZut LI60Si tigen. scieled atungiene wadigat He deed eet 3.0 grams. 
ABU Tis scien asc thors teak den Nee Ra RAR DRM wheal 8 o.8 gram. 

Gly cerniees: esa eSes asc ae shes sa ga eee 250.0 grams. 
Methyl alcohol........... 0.0 c cece ees Sacwane 250.0 grams. 


. Dissolve the powdered dyes in the glycerin at 60° C.; then add 
the methyl alcohol previously heated to the same temperature. 
After mixing, let it stand 24 hours at room temperature, and 


44 * BACTERIOLOGY 


filter. To stain, mix one drop of this solution with 1 c.c. of water 
and immerse the film, previously fixed, for 15 minutes to 24 hours. 

Direct Preparation of Romanowsky Stains —In a study of the 
essential constituents of the Romanowsky stain, MacNeal! 
found both methylene azure and methylene violet to be present 
and participating in the nuclear staining. The preparation of 
solutions directly from the-pure dyes, methylene azure, methylene 
violet, methylene blue and eosin, has been recommended as the 
best manner of preparing these staining solutions, as the propor- 
tion of the various constituents may be varied at will to obtain 
various kinds of differentiation. As a routine blood stain for 
study of leukocytes and staining of hematozoa, the following is 
recommended: 


Solution A. 
Meth y lemme az urescsic) ai; canaraiinincuaien tives uae tania ete des 0.3 
Methylene violet (Bernthsen’s, insoluble in | soos 0.2 
Methylene blue, medicinal...............000cceeeeeeee I.0 
Methyl alcohol, pure.......... 0.0 ccc cece ene eee 500.0 
Solution B. 
Eosin, yellowish, water soluble..................0000005 1.0 
Methyl alcohol, Ure x: 9 snus ses tage aie be aehenase bere 500.0 


These solutions keep for at least a year. They are mixed in equal 
parts and filtered to prepare the actual staining solution, which 
keeps for a few weeks. This final mixture is employed in the same 
manner as Leishman’s stain. It keeps for a few months. 
Method of Staining Cover-glass Preparations.—(a) A smear 
preparation of bacteria having been made and fixed in the manner 
above described, and a watery solution of either fuchsin, gentian 
violet or methylene blue having been prepared, the cover-glass 
is to be dropped into a dish containing the dye, or the dye may 
be dropped upon the cover glass held in the forceps. 
(0) Allow the stain to act for about thirty seconds. 
(c) Wash in water. 
(d) Examine with the microscope in water directly or after . 
drying and mounting in Canadian balsam. 
Journ. Infectious Diseases, Vol. III, 1906, pp. 412-433. 


THE MICROSCOPE AND MICROSCOPIC METHODS 45 


The rapidity and intensity of staining may be increased by 
warming the solution slightly. The bacteria will usually appear 
more distinct if, directly after pouring off the stain, the prepara- 
tion is rinsed for a few seconds in 1 per cent solution of acetic 
acid, and then thoroughly washed in water. The acetic acid 
solution serves to remove in a measure any color which has 
been imparted to the background, and which is undesirable. 

Preparations that are mounted at first in water may be made 
permanent by moistening the edge of the cover-glass so that it 
may be easily removed from the slide, then drying and mounting 
in Canada balsam. Cover-glass preparations which have been 
stained are.examined with the oil-immersion objective, employ- 
ing the plane mirror, having the iris diaphragm open and the 
condenser close to the lower surface of the glass slide. The 
purpose is to obtain the most intense illumination possible over 
a small field. . 

Gram’s Method.—Cover-glass preparations, having been pre- 
pared and fixed in the usual manner (see page 38), are stained 
as follows: 

(a) Stain in carbol gentian violet or in malnepate gentian 
violet solution, from two to five minutes. The intensity of the 
stain may be increased by warming slightly. 

(b) Gram’s-solution, one and one-half minutes: 


3 MOGING 2 senda cs Med day Ree bape ee EE pea Be ing I gram. 
Potassium iodide. 2 ...0ccccke te cena beet ee bea g tea 2 grams. 
Wate iia ahs dr aia ean M SARA S Ah.guiors Rateneuymems Geen 300 C.C. 


In this solution the preparation becomes nearly black. 

(c) Wash in alcohol repeatedly; the alcohol becomes stained 
. with clouds of violet coloring matter; the alcohol is used as long 
as the violet color continues to come away, and until the prepara- 
tion is decolorized or has only a faint steel-blue color. 

(d) When desired, the specimens may be stained, by way of 
contrast, with a watery solution of Bismarck brown, dilute fuchsin, 
safranin or eosin. 


46 BACTERIOLOGY 


(e) Wash in water, and examine either in water directly or 
after drying and mounting in Canada balsam. Gram’s method 
and its modifications should not be regarded as absolute means 
of distinguishing between Gram-positive and Gram-negative bac- 
teria in every case, as much depends upon the condition of the 
bacteria, and very much upon the technic of staining. When the 
Gram stain is used for diagnosis, it is well to put a smear of a 
known Gram-negative and a smear of a known Gram-positive 
organism on the same slide or cover-glass along with the un- 
known, and subject them all to the same technic. 

Some bacteria that are stained by Gram’s method: 

Staphylococcus aureus, 

Streptococcus pyogenes, 

Micrococcus lanceolatus (of pneumonia), 
Micrococcus tetragenus, 

Bacillus of diphtheria, 

Bacillus of tuberculosis, 

Bacillus of leprosy, 

Bacillus of anthrax, 

Bacillus of tetanus, 

Bacillus welchii (aérogenes capsulatus), 
Ray fungus of actinomycosis. 

Of these the tubercle bacillus and the bacillus of leprosy 
require a much longer exposure to the stain than other bacteria 
in the list. 

Some bacteria that are not stained by Gram’s method: 

Gonococcus, 

Diplococcus intracellularis (meningitidis), 
Micrococcus melitensis, 

Bacillus of chancroids (Ducrey), 

Bacillus of dysentery (Shiga), 

Bacillus of typhoid fever 

Bacillus coli, ‘ 

Bacillus pyocyaneus, 

Bacillus of influenza, 


THE MICROSCOPE AND MICROSCOPIC METHODS 47 


Bacillus of bubonic plague, 

Bacillus of glanders (Bacillus mallei), 
Bacillus proteus, 

Spirillum of Asiatic cholera, 
Spirillum of relapsing fever. 

Staining of Acid-proof Bacteria—A very large number of 
methods have been proposed for staining the tubercle bacillus, 
all of which depend upon the principle that, after adding to 
solutions of aniline dyes certain substances, like aniline water, 
carbolic acid, or solutions of ammonia or soda, the tubercle bacillus 
is stained with great intensity, and gives up its stain with difficulty. 
Solutions of acids will remove the stain from all parts of the prepa- 
ration excepting from the tubercle bacilli, which retain the dye, 
having once acquired it. The rest of the preparation may now 
be given a different color—contrast-stain. 

Bacilli that resist decolorization by acids are called acid-proof 
or acid-fast. 

Some acid-proof bacteria : 

Bact. tuberculosis, 

Bact. lepre, 

Bact. smegmatis, 

Grass bacillus of Moeller, 

Butter bacillus of Rabinowitsch, 

Certain streptothrices, 

Certain bacilli common in the feces of cattle, 
Certain bacteria found in distilled water, 
Spores of many bacteria. 

Occasionally other bacteria, micrococci and horny epithelial 
cells are imperfectly decolorized, but their forms distinguish 
them from tubercle bacilli. Minute crystalline needles which 
have a shape like that of bacilli, are often encountered in sputum, 
but%their nature will be recognized after a little practice. 

The stain for acid-proof bacteria is most frequently used for 
specimens of ‘sputum from cases of suspected pulmonary tubercu- 
losis; it may be applied to other fluids and secretions equally 


48 BACTERIOLOGY 


well. It is not reliable, however, when applied to milk, as the 
oil present in milk interferes with its operation, and milk and 
its products quite often contains other acid-proof bacilli. The 
smegma of the external genitals also frequently contains acid- 
proof bacilli that are not tubercle bacilli. On this account all 
fluids and discharges from the genito-urinary tract need to be 
examined with particular care not to confuse tubercle bacilli 
with smegma bacilli. Too much reliance should not be placed 
on the possibility of distinguishing between tubercle and smegma 
bacilli by decolorizing in alcohol. In doubtful cases an animal 
should be inoculated. 

Patients should be given minute instructions concerning the 
collection of sputum. The bottle used- should be new, wide- 
mouthed, clean, and kept tightly stoppered with a clean cork. 
The patient should be cautioned against allowing the expectora- 
tion to get on the outside of the bottle. Probably whatever 
risk is incurred by those who examine sputum comes chiefly 
from the outside of the bottle having been soiled with sputum 
containing tubercle bacilli. It is well to disinfect the exterior 
of the bottle when it is received at the laboratory. Often little 
white particles may be seen ficating in the mucous portions of 
the sputum. These particles should be selected for the investiga- 
tion, and may be spread in a thin film on the cover-glass with the 
platinum wire, which is sterilized in the flame before and after 
using. The selection of the little white particles will be faciliated 
if the sputum be poured into a clean glass dish, which may be 
placed on a black surface. A form of porcelain dish is furnished 
by dealers, the bottom of which is black, and which is convenient 
for these manipulations. The smears may be made moderately 
thick as a larger amount of sputum may thus be examined in a 
short time. Uniform thickness is difficult to obtain and is not 
absolutely essential. It is hardly necessary to observe that the 
operator must be scrupulously careful not to contaminate the ma- 
terial under examination with any kind of extraneous matter 
The cover-glasses and slides which are used should be new, and 


THE MICROSCOPE AND MICROSCOPIC METHODS 40 


should have been cleaned with bichromate of potassium and sul- 
phuric acid (see page 37). When the work is completed, the 
bottle containing the sputum should be sterilized by steam or 
boiling. 

Method for staining the tubercle bacillus: 

(a) The cover-glass or slide preparation is made, dried, and 
fixed by passing through the flame three times. 

(b) The cover-glass, held in forceps or in a watch-crystal is 
covered with steaming carbol-fuchsin for five minutes. If a 
slide is employed it may be conveniently stained in the Krauss 
staining dish, being turned face downward. 

(c) Wash in water. 

(d) Wash in alcohol containing 3 per cent of hydrochloric acid 
one minute, or longer if necessary to remove the red color. 

(e) Wash in water. 

(f) Stain with methylene-blue solution (see page 42) thirty 
seconds. 

(g) Wash in water. 

(h) Examine in water directly, and after drying and mounting 
in Canada balsam. If the preparation has been made on a slide 
it may be dried and examined directly in cedar oil with the 14» in. 
objective. When the preparation is mounted in water, tubercle 
bacilli may be obscured by refraction in the thicker portions of 
the smear. Tubercle bacilli take a brilliant red color; other bac- 
teria and the nuclei of cells are stained blue. 

Of the numerous methods of staining tubercle bacilli only a few 
others can be mentioned. Aniline-water fuchsin,.aniline-water 
gentian violet, or carbol-fuchsin may be used. The intensity of 
the stain must then be increased by warming the preparation till 
it steams or boils, then allowing the warm stain to act on the 
specimens for from three to five minutes; the preparation may also 
be left in the cold stain over night. Decolorization may be effected 
with a 25 per cent solution of sulphuric acid used till the red color 
disappears, or a 30 per cent solution of nitric acid, which operates 
very rapidly. If the red color persists after washing in water, 

4 


- 


50 BACTERIOLOGY 


dip in the acid again. After either acid the preparation is to be 
washed in alcohol until the last trace of the stain has been removed. 
An excellent decolorizing agent is a 3 per cent solution of hydro- 
chloric acid in alcohol, used for about a minute. The contrast 
stain may be omitted entirely if it is desired. A suitable contrast 
stain after fuchsin staining is a solution of methylene blue; after 
gentian-violet staining, safranin. 

Those who have had experience in staining tubercle bacilli 
sogn discover that the bacilli exhibit some differences in their 
resisting power to strong acids. One encounters occasionally 
bacilli that are perfectly stained side by side with others that are 
more or less completely decolorized. These facts show the ne- 
cessity of practice with any method, and of exercising caution 
and judgment in making a diagnosis where the number of bacilli 
happens to be scanty. If tubercle bacilli are not found in the 
first preparation, other preparations should be made. Some- 
times a large number of cover-glasses must be examined. 

Various expedients have been devised to concentrate tubercle 
bacilli when only a small number may be present in a sample of 
sputum. Antiformin (a preparation of chlorinated sodium hydrox- 
ide) has been employed for this purpose. The following method 
is that of Williamson.1 The sputum is measured and transferred 
to a clean flask of resistant glass. An equal volume of 50 per 
cent antiformin is added, mixed with the sputum, and the mixture 
brought to a boil over the flame. This dissolves the sputum 
promptly. The.material is then cooled and to each to c.c. of 
material in the flask, 1.5 c.c. of a mixture of chloroform, one part, 
and alcohol, nine parts, is added. The mixture is thoroughly 
shaken. Asa result the tubercle bacilli imbibe some of the chloro- 
form and become heavier. The material is next centrifugalized 
at high speed for 15 minutes, which separates it into three layers, 
antiformin above and chloroform below with the layer of sediment 
between the two. This layer is removed and mixed with egg albu- 
men (egg albumen + 0.5 per cent carbolic acid) on a slide and then 

1 Williamson, Journ. A. M. A., Apr. 6, 1912, Vol. LVIII, p. 1005-07. 


THE MICROSCOPE AND MICROSCOPIC METHODS 51 


spread into a smear between two slides. The smears are then 
dried and stained in the usual way. Instead of using albumen 
to fix the sediment to the slide, it is convenient to save some 
of the original sputum and mix it with the sediment for this 
purpose. 

Staining of Spores.—The method is applicable to cover- 
glass preparations which may be prepared in the usual way from 
material supposed to contain spores. 

(a) After drying the smear on the cover-glass, fix it with heat- 
by passing through the flame three times. 

(b) Float the cover-glass face downward on the surface of 
steaming hot carbol-fuchsin or aniline-water fuchsin for three to 
five minutes. 

(c) Wash in 3 per cent hydrochloric acid alcohol one minute, 
or less. 

(d) Wash in water. 

(e) Stain with watery solution of methylene blue half. a 
minute. 

(f) Wash. 

(g) Dry. 

(h) Balsam. 

The spores are intensely stained by the fuchsin. The stain 
is removed from*everything except the spores by the acid alcohol. 
The methylene-blue solution stains the bodies of the bacteria, 
the spores remaining brilliant red. There are various other 
methods for staining spores, but this procedure usually gives 
good results. The principle is the same as in staining the tubercle 
bacillus, except that more pains are needed to impregnate spores 
with the dye. 

When it fails, the cover-glass preparation may be treated by 
Moeller’s method previous to staining. After fixation, the prep- 
aration is immersed in chloroform for 2 minutes, drained and 
dried in the air. It is then immersed in 5 per cent chromic 
acid for 2 minutes, washed thoroughly in water, and stained 


as above described. 


52 BACTERIOLOGY 


Staining of Capsules—The capsules which many bacteria 
possess, appear to be made of some gelatinous substance, which 
is difficult to stain. 

Method of Hiss.—(a) Cover-glass preparations are made in 
the ordinary way and fixed in the flame as soon as dry. 

(b) Use the following stain, heated till it steams: 


Saturated alcoholic solution of gentian violet or fuchsin...... 5 c.c. 
Distilled: Water cccsunscneeuia ch Heed anima Yee Galena aero 95 C.c. 


(c) Wash in 20 per cent solution of cupric sulphate crystals. 

(d) Dry and mount in Canada balsam. 

The method of Hiss is recommended to be used for bacteria that 
have been cultivated on serum-agar with 1 per cent of dextrose. 
This method gives excellent results with preparations of capsulated 
bacteria in smears of animal tissues. 

Method of Huntoon!.—In this method two solutions are re- 
quired. 

Solution 1—Sift 3 grams nutrose into 100 c.c. distilled water 
and steam for one hour. Then add 5 c.c. of 2 per cent. aqueous 
solution of carbolic acid. Allow the sediment to settle and employ 
the supernatant liquid as the diluent. 

Solution 2.—Mix the following, 100 c.c. of 2 per cent aqueous 
carbolic acid; 0.25 to 0.50 c.c. of concentrated lactic acid; 1 c.c. 
of 1 per cent acetic acid; 1 c.c. of saturated alcoholic fuchsin; 
and 1 c.c. of old carbol-fuchsin solution. 

Solution 1 is placed on the slide and the bacteria are emulsified 
in it and then spread with the platinum loop. When dry the film 
is covered at once with solution 2 for thirty to fifty seconds. Wash 
quickly in water, dry and examine. . 

Staining of Flagella.—Flagella are among the most difficult 
of all objects to stain. The best-known method is that of Léffler. 
It is important to use young cultures (4 to 10 hours old), preferably 
-on agar. 

(a) A small amount of the growth is gently mixed with a 

* Huntoon, Journal of Bacteriology, May, 1917, Vol. II, p. 241. 


THE MICROSCOPE AND MICROSCOPIC METHODS 53 


large drop of distilled water on a clean slide, so that the water is 
made very faintly cloudy. From the top of this drop one or 
two transfers are made to a second drop with a small platinum 
loop. From this second drop a loopful is transferred to a per- 
fectly clean (flamed) cover-glass, spread with minimum manipu- 
lation and dried quickly, high over the flame. 

(b) After drying, fixation is effected by passing through the 
flame three times, holding the cover-slip between the thumb and 
fore finger to avoid overheating. 

(c) The essential point in this method is the use of a mordant 
as follows: 


Tannic acid, ro per cent solution................. ee 20 C.C. 
Saturated solution of ferrous sulphate.................. 4C.C. 
Saturated alcoholic solution of fuchsin................. I CC. 


This solution should be freshly prepared from pure substances, 
and should be filtered at once after mixing. It may deteriorate 
in a few hours but sometimes keeps for a few days or weeks. 
A few drops are placed on the cover-glass, or the cover-glass is 
placed, face down, in a dish containing the mordant; it is then 
left for one to five minutes, the mordant being heated until it 
steams. 

(d) Wash in water. 

(e) Stain with aniline-water fuchsin, or carbol-fuchsin, hot, 
for one to two minutes. 

(f) Wash in water. 

(g) Dry. | 

(h) Mount in Canada balsam. 

Another and very valuable method is that of Van Ermengem. 

(a) Make and fix cover-glass preparations as in the preceding 
method. 

(b) Use the following mordant for one-half hour at room tem- 
perature or for five minutes at 50° to 60° C. 


Osmic acid 2 per cent solution..........0... 0000s e eee eee cia 
Tannic acid 10 to 25 per cent solution............--...-+0.. 2 


(c) Wash carefully in distilled water and then in alcohol. 


54 BACTERIOLOGY 


(d) Place for a few seconds in a 0.25 to 0.50 per cent solution of 
nitrate of silver—‘the sensitizing bath.” 

(e) Without washing transfer to the ‘‘reducing and reinforcing 
bath”’: 


Galle acid: sienicaks hades Tala S RD aide dainhaass tes. 5 grams. 
Pannicacid paws caw sey goreceoeeep wee cern 3 grams. 
Fused potassium acetate............. peeises segs Io grams. 
Distilled Watetisn.cescsuveiveasaarasscieenanen awe 350 C.C. 


(f) After a few seconds, replace the preparation in the nitrate 
of silver solution, in which it is kept constantly moving, till the 
solution begins to acquire a brown or black color. 

(g) Finally wash in distilled water, dry, mount in Canada 
balsam. It is difficult to avoid the formation of precipitates; 
otherwise the results of this method are usually good. 

Wet Fixation of Protozoa.—The fluid containing the protozoa 
is spread on a cover-glass or slide and immediately dropped upon 
a solution of the fixing agent, commonly sublimate alcohol heated 
to 60° C. This is prepared by mixing saturated aqueous solution 
of mercuric chloride, 100 c.c, with absolute alcohol, 50 c.c., 
and acetic acid, 5 drops. After a few minutes the preparation 
is carefully washed in water, and passed through graded alcohols 
toharden. It may then be stained, dehydrated in graded alcohols, 
cleared in xylol and mounted in balsam. The preparation should 
not be allowed to dry at any stage of the process. 

Heidenhain’s Iron Hematoxylin—The preparation to be 
stained by this method should be fixed in mercuric chloride or 
alcohol. The stain is prepared by dissolving hematoxylin crys- 
tals, 1 gram, in hot absolute alcohol 10 c.c., and then adding dis- 
tilled water 90 c.c. This solution is allowed to stand in an open, 
cotton-plugged bottle for about four weeks, and it is then diluted 
with an equal volume of water before using. The iron solution 
is made by dissolving 2.5 grams of ferric ammonium sulphate 
(lavender-colored crystals) in 100 c.c. of distilled water. The 
preparation to be stained is first soaked in the iron solution for 
four to eight hours, then rinsed and immersed in the hematoxylin 


THE MICROSCOPE AND MICROSCOPIC METHODS 55 


for twelve to twenty-four hours. It is again rinsed and now 
differentiated by immersion in the iron solution until black clouds 
cease to be given off. When the desired differentiation has 
been obtained the preparation is washed, dehydrated by passing 
through graded alcohols, and absolute alcohol, cleared in one 
and mounted in balsam. 

Preparation and Staining of Blood Films.— Blood films are 
best made on clean, flamed slides. A small drop of fresh blood is 
received on the surface of one slide near one end. The end of 
another slide is applied to the first at an acute angle so that the 
blood spreads laterally in the angle between the two slides. The 
second slide is then pushed along the surface of the first with the 
blood following it in the angle. The thickness of film may be 
regulated by varying the size of angle between the two slides as 
well as by the speed of movement. 

For staining blood films, either Leishman’s or Giemsa’s stain 
or some modification of them should be used as a general rule. 
After fixation in absolute alcohol, blood films may bé stained with 
Léffler’s methylene-blue or by Gram’s method. 

Staining Bacteria in Tissues.—Pieces of organs about 1 cm. 
in thickness may be taken. Alcohol is the best agent for preserv- 
ing them. The hardening will be completed in a few days. It 
is best to change the alcohol. The amount of the alcohol must 
be twenty times the bulk of the tissue to be preserved. 

Ten parts of the standard 40 per cent solution of formalde- 
hyde, with 90 parts water make a good mixture for fixation; after 
twenty-four hours change to alcohol. 

Imbedding in Collodion or Celloidin.—From absolute alcohol 
the pieces of tissue are placed in equal parts of alcohol and ether 
twenty-four hours; thin collodion (114 per cent.), twenty-four 
hours; thick collodion of a syrupy consistency (6 per cent) twenty- 
four hours. The specimen is laid upon a block of wood and 
surrounded by thick collodion, and then inverted in 70 percent 
alcohol. The collodion makes a firm mass, surrounding and per- 
meating the tissue, and permits very thin sections to be cut. 


56 BACTERIOLOGY 


The soluble cotton sold by dealers in photographer’s supplies serves 
as well as the expensive preparation known as celloidin. To 
make collodion, dissolve it in equal parts of alcohol and ether. 
Soluble cotton is also called pyroxylin, and is a kind of gun-cotton. 

Imbedding in Paraffin.—(a) Pieces of tissue 2 to 3 mm. thick 
which have already been fixed in alcohol or formaldehyde aré to 
be placed in absolute alcohol for twenty-four hours. 

(0) In pure xylol one to three hours. 

(c) Ina saturated solution of paraffin in xylol one to three hours. 

(d) In melted paraffin having a melting-point of 50° C., which 
requires the use of a water-bath or oven, one to three hours. The 
xylol must be entirely driven off, and the tissue thoroughly 
infiltrated. 

(e) Change to fresh paraffin for one hour. 

(f) Finally, place the tissue in a small dish or paper box and 
pour the melted paraffin about it. Harden as quickly as possible 
with running water. It is important to fix the piece of tissue 
in a suitable position, if the position is of importance, before 
pouring in the melted paraffin. Sections of exquisite thinness 
may now be cut. The knife need not be wet. Paraffin im- 
bedding is especially desirable when serial sections are to be made. 

In order to mount the sections, proceed as follows: 

(a) Place the sections on water in a porcelain capsule. 

Warm slightly, when the sections will flatten. Smear the surface 
of a slide with a very thin layer of Mayer’s glycerin-albumen 
mixture. Dip the slide under the sections; lift them; and then 
drain off the water, leaving the sections in their proper positions. 
Let them dry for some hours in the incubator, and they will be 
firmly fastened to the slide. 


GLYCERIN-ALBUMEN MIXTURE (MAYER) 


Equal parts of white of egg and glycerin are thoroughly mixed, and then filtered. 
Add a little gum-camphor to preserve. 


(b) Dissolve out the paraffin in one of the numerous solvents 
(xylol, a few minutes). 


THE MICROSCOPE AND MICROSCOPIC METHODS 57 


(c) The xylol should be washed off with absolute alcohol, 
and then 70 per cent alcohol and finally distilled water. 

(d) The section is stained. 

(e) Dehydrate in absolute alcohol. 

(f) Clear in xylol. 

(g) Mount in balsam. 

Section Cutting.—Cutting is best done with an instrument 
‘called a microtome. The tissues may be imbedded in collodion 
or paraffin; or when they have been hardened with formaldehyde 


aaaae 


Fic. 27.—Automatic laboratory microtome. 


they may be cut after freezing. Bacteria stain admirably in 
frozen sections. For routine work collodion imbedding will be 
found as convenient a process as any. Paraffin imbedding gives 
the thinnest sections. 

A microtome consists of a heavy, sliding knife-carrier, which 
moves with great precision on a level, and of a device for elevating 
the object which is to be cut, any desired distance after each ex- 
cursion of the knife. The thickness of the section will be the 
distance which the object is elevated. The knife is kept wet 
with alcohol during the cutting of collodion sections, otherwise 
it is left dry. The microtome is usually provided with a special 
form of knife. A razor will serve nearly as well, after having 


58 BACTERIOLOGY 


had the lower side ground flat. If a razor is used, a special form 
of razor-holder must be attached to the microtome to receive the 
razor. Above all, it is necessary that the knives should be kept 
in good condition. Only occasionally will they need honing, using 
a fine water-stone or Belgian hone. The movement in honing 
should be from heel to toe, always placing the back of the knife 
next the hone when turning. The knife should be stropped fre- 
quently. The leather of the strop should be glued to a strip of: 
wood to make a flat surface. The movement in stropping should 
be from toe to heel. Sections should be cut to a thickness of not 
more than 25 4. Thinner sections (5 to 10 u) are to be desired. 

Staining of Section.—A watery solution of one of the aniline 
dyes is used—fuchsin, gentian violet or methylene blue—made 
by adding a few drops of the alcoholic solution to a dish filled 
with water. Léffler’s solution of methylene blue serves very 
well. 

By this process most bacteria are stained; also the nuclei of 
cells; frequently, also certain granules contained within some cells 
which may easily be mistaken for bacteria by the inexperienced 
(basophilic granules). 

(a) Place the section in the staining solution from two to five - 
minutes. 

(b) Wash in water. 

Place in a watery soluticn of acetic acid, 1 per cent, for: 
one minute. 7 

(d) Alcohol, one to two minutes; change to absolute alcohol. 
Touch the sections to blotting-paper to remove the superfluous 
alcohol. 

(e) Xylol until clear; xylol is to be preferred to other clearing 
agents, like oil of cloves, most of which slowly remove aniline 
colors. It has the disadvantage of not clearing when more thana 
trace of water is present; dehydration in alcohol must, therefore, 
be complete. The section should be removed from the xylol as 
soon as it is cleared; otherwise wrinkling occurs. 

(f) The section is placed upon a glass slide; a drop Canada 


THE MICROSCOPE AND MICROSCOPIC METHODS 59 


balsam is placed upon it and then a cover-glass. The Canada 
balsam should be dissolved in xylol. 

The section is to be manipulated with straight or bent needles. 
The removal from xylol to the glass slide is managed best with a 
spatula or section-lifter. 

The above statements apply to frozen sections or to sections 
imbedded in celloidin. Paraffin sections are preferably attached 
to the slide with glycerin-albumen. The different steps in the 
process follow in the same order. The stain may be poured on 
the slide, or the slide may be placed in a large dish full of staining 
fluid. (See page 44.) Celloidin sections may also be stained on 
the slide. If the section be well spread and flattened thoroughly 
with blotting paper, it will usually adhere to the slide, and is less 
likely to wrinkle. It must not be allowed to dry. 

Gram’s Method may be applied to the staining of sections 
of tissues as well as to smears upon cover-glasses. 

(a) Place the section in aniline-water gentian violet, one to 
five minutes. 

(6) Rinse briefly in water. 

(c) Iodine solution (see page 45), one and one-half minutes. 

(d) Alcohol, until decolorized to a faint blue-gray. 

(e) Xylol. 

(f) Mount on a slide in balsam. 

Weigert’s Modification of Gram’s Method, or Weigert’s Stain 
for Fibrin.—(a) Place the section in aniline-water gentian violet 
solution, five minutes or more. 

(b) Wash briefly in water. 

(c) Place the section upon a slide by means of a section lifter; 
having straightened it carefully, absorb the water with blotting- 
paper. ‘ 

(d) Gram’s solution (see page 45) one to two minutes. 

(e) Absorb the iodine solution with blotting-paper. 

(f) Add aniline oil, removing it from time to time with blotting- 
paper, and adding fresh aniline oil until the color ceases to come 
away. (Aniline oil serves in this connection both to decolorize 


60 BACTERIOLOGY 


and to dehydrate. It absorbs the water rapidly and efficiently. 
However, on account of its decolorizing tendency, it must be re- 
moved before the specimens can be mounted permanently.) 

(g) Add xylol; remove it with blotting-paper; and add fresh 
xylol several times, in order to extract the last trace of aniline oil. 

(hk) Mount in Canada balsam. 

This method is more convenient for the staining of sections 
than the Gram method. The results, however, are essentially 
the same as far as the bacteria are concerned; fibrin and hyaline 
material are stained blue, bacteria violet. It is often impossible 
to decolorize the nuclei. completely without decolorizing the 
bacteria also. The parts of the nuclei which remain stained 
often present pictures that resemble bacteria, and which may 
lead to error if not recognized. Basophilic granules also retain 
the stain, as do the horny cells of the epidermis. These remarks 
apply also to Gram’s method, except as regards fibrin. Very 
beautiful preparations can be obtained according to this or the 
Gram method when the sections have previously been stained 
in carmine; the nuclei will then be colored red, bacteria violet. 

Tubercle bacilli may be stained in sections as follows: 

(a) Use carbol-fuchsin, or aniline-water gentian violet for 
one-half to two hours with very gentle warming, or over night 
without warming. 

(b) Wash in water. 

(c) Decolorize with some one of the decolorizing agents men- 
tioned in connection with the staining of tubercle bacilli in cover- 
glass preparations, preferably 3 per cent hydrochloric-acid alcohol. 
Decolorization must be continued’ until the red color has dis- 
appeared, which requires one-half to several minutes. 

(d) Wash in alcohol. 

(e) Wash in water. 

(f) Use hematoxylin as a contrast-strain for fuchsin prepara- 
tions, and carmine for gentian violet preparations. (It is bet- 
ter to stain with carmine first of all and before staining the bacilli. 
The carmine is not affected by the subsequent treatment.) 


THE MICROSCOPE AND MICROSCOPIC METHODS 61 


(g) Wash in water. 
(h) Alcohol. 
(z) Xylol. 
(7) Balsam. 
Nuclear stains, which may be used as contrast-stains for 
sections: 
DELAFIELD’s HEMATOXYLIN 


‘Hematoxylin Crystals aya scauaersema coadmennad asks 4 grams. 

SAlCOhO! ssis.scapatact sidered 6 ew LEK gual dimthashoadbamantee 25 C.C. 
Ammonia alum...............00 00. cece cece neces 50 grams. 
Wallet conte ane ttd.aid-actacanti linea oes Ghee pie aaaneeetetias 400 C.C. 
GLY CCE c Sserats tated wine Spa's Ha Bema x lee or SS 100 C.Cc. 
Methyl alcohol...... i aif Big-bad hha Fasct Sees domes ue Gabe 100 C.c. 


Dissolve the hematoxylin in the alcohol, and the ammonia 
alum in the water. Mix the two solutions. Let the mixture 
stand four or five days uncovered; it should have become a deep 
purple. Filter and add the glycerin and the methyl alcohol. 
After it has become dark enough, filter again. Keep it a month 
or longer before using; the solution improves with age. At the 
time of using, filter and dilute with water as desired. | 


LITHIUM-CARMINE (ORTH) 


Carmine. ick dcae cesar ioe den de Maina ae 2.5 grams. 
Saturated watery solution of lithium carbonate.... 100.0 c.c. 


Add a few crystals of thymol. The carmine dissolves readily 
in the lithium carbonate solution. Filter the stain at the time of 
using. Sections are to be left in the stain five to twenty minutes. 

Sections stained in carmine are placed directly in acid alco- 
hol ( 1 part hydrochloric acid, 100 parts 70 per cent alcohol) for 
five to ten minutes. They acquire a brilliant scarlet color. 
When used as a contrast-strain for tissues containing bacteria, 
it is best to use it before staining the bacteria, which might be 
decolorized by the acid alcohol. 


CHAPTER II 


STERILIZATION—D ISINF ECTION—ANTISEPSIS— 
FOOD PRESERVATION 


Definitions.—By sterilization is meant the killing or the re- ° 
moval of all micro-organisms in or on a body or substance. Dis- 
infection has a somewhat analogous signification, but denotes 
the destruction or removal of infectious microbes, and this may 
or may not be accomplished without complete sterilization, 
according to the nature of the particular case in hand. Anti- 
sepsis means the inhibition of growth of micro-organisms with- 
out ordinarily killing or removing them, and is especially applied 
to the checking of microbic activity in wounds and the effects 
produced thereby (sepsis). Food preservation involves similar 
principles, depending upon the prevention of microbic activity 
in dead organic matter either by sterilization or by the presence 
of inhibitive substances, similar to antiseptics, but in this instance 
called preservatives. 

In connection with sterilization we shall consider those agents 
which remove or destroy a part of the microbic flora without 
producing complete sterility, as well as the methods which insure 
complete sterilization. A few examples of each general class 
will be considered. 

Physical SterilizationAmong the physical means by which 
sterilization may be accomplished, those which are merely me- 
chanical may be mentioned first. The removal of microbes from 
an infected surface by washing them away is a method of wide 
application., Complete sterility may sometimes be attained in 
this way. In ordinary disinfection of woodwork, walls and 


floors, or of the hands, mechanical cleaning is of primary impor- 
62 


STERILIZATION—-ANTISEPSIS—-FOOD PRESERVATION 63 


tance, even though it does not insure complete sterilization. 
The process removes not only many of the bacteria, but also 
much other material which serves to protect them and even to 
furnish food for their development. Another mechanical method 
is that of comminution, actual crushing of the bacterial cells. 
It is of very narrow application and not to be relied upon. High 
pressures have been employed to destroy bacteria, but hydrostatic 
pressure of even 1000 atmospheres does not produce complete 
sterilization. Sedimentation is a method of primary importance, 
especially in the removal of suspended bacteria from the atmos- 
phere. It also operates to remove a large proportion of the 
bacteria from ‘drinking water when stored in suitable reservoirs. 
Filtration of fluids is an important means of sterilizing them. 
Air may be sterilized by drawing it slowly through a sufficient 
layer of cotton. Water becomes bacteria-free as it filters through 
the soil, so that waters from the depths of the earth are sterile. 
Liquids are commonly sterilized in the laboratory by forcing them 
through a layer of unglazed porcelain (Pasteur-Chamberland filter) 
or through a compact wall of diatomaceous earth (Berkefeld 
filter). Liquids rich in bacteria, such for example as cultures in 
broth, may be rendered bacteria-free in this way. These filters 
have also been employed for the sterilization of drinking water, 
but their use for this purpose requires intelligence and care, and 
when carelessly employed they are worse than useless. 

Desiccation is destructive to many microbes, especially those 
which do not form spores. The germs of Asiatic cholera are dead 
in a few hours after complete drying. The spores of the anthrax 
bacillus on the other hand remain alive for at least ten years after 
drying. Most bacteria resist drying long enough so that they 
may be transferred by air currents as dust and still be capable of 
growth. 

Light is injurious to bacteria and direct sunlight is rapidly 
fatal to them, even in spore form. Light seems to act by pro- 
ducing powerful chemical germicides, probably organic peroxides, 
in the medium surrounding the bacteria. Such substances are 


64 BACTERIOLOGY 


known to be produced under these circumstances. They rapidly 
decompose. 

Cold appears to be fatal to some pathogenic forms, and a con- 
siderable percentage of the bacteria in a culture are usually killed 
by freezing. Cultures cannot be completely sterilized even by 
exposure to the temperature of liquid air. Cold is therefore not 
to be regarded as an efficient germicide, although it may com- 
pletely check the growth of bacteria. 

Heat is the most important of the physical means and doubtless 
the most important of all means of destroying bacteria. Its 
value as a purifying agent was recognized among the ancients. 
Heat is applied under conditions insuring the presence of liquid 
water, so-called moist heat, and in the absence of water, so-called 
dry heat or hot-air sterilization. The most reliable methods of 
sterilization by dry heat are those which accomplish the combus- 
tion or destructive distillation of organic matter’ in general. 
Actual combustion of clothing and bedding, and even of houses 
‘has been resorted to in the past as a method of disinfection, Heat- 
ing to redness in the naked flame is the routine method of steriliz- 
ing our platinum wire, and glass articles, such as capillary pipettes, 
cover-glasses and slides are commonly sterilized in the flame. 
Flaming may even be employed for sterilization of surgical instru- 
ments in an emergency, although such treatment quickly destroys 
steel instruments. Sterilization.of large objects and of combus- 
tible material by dry heat is generally accomplished in an oven or 
hot-air sterilizer. The common laboratory sterilizers are boxes of 
sheet iron with double walls, with air space between to allow the 
hot gases from the flame completely to surround the inner com- 
partment. The door, which occupies one full side, is usually 
double. A tubulation through the top allows a thermometer to be 
inserted into the interior so that the temperature may be read off 
at any time. Even the best hot-air sterilizers fail to give an even 
temperature throughout the interior, so that the thermometer 
bulb at one corner cannot be implicitly relied upon to record the 
temperature of other parts. Ordinarily a temperature of 150° C. 


STERILIZATION—ANTISEPSIS—-FOOD PRESERVATION 65 : 


for one hour, 170° C. for 30 minutes, or 200° C. for one minute will 
kill all bacteria. Such exposure browns cotton of good grade only 
slightly. One fallacy in hot-air sterilization needs to be guarded 
against. Glassware and other apparatus must be dry before it 
is put into the oven to sterilize. A tube containing water may be 
left in the oven until the thermometer records a temperature of 
200° C. in the uppef corner of the sterilizer, and subsequently the 


a 


unl il. 


— 

aM | 

| a4 | 
fA 


Fic. 28.—Hot-air sterilizer. 


tube may be removed from the oven with the most of the water still 
in it. Hot-air sterilization is employed for glassware, tubes with 
cotton plugs, granite-ware, stone-ware, and for metals not injured 
by heat. 

Moist heat or heat in the presence of liquid water must be used 
whenever drying is to be avoided, especially in the sterilization of 
culture media and various solutions. It is employed as continu- 
ous sterilization at a single exposure and as discontinuous ster- 


ilization, heating for a short time on several consecutive days. 
5 


66 BACTERIOLOGY 


The temperature employed varies according to the effect desired. 
A temperature of 60° C., maintained throughout a water liquid 
for twenty minutes will kill most vegetative bacteria, and practi- 
cally all pathogenic bacteria which do not form spores. Such 
partial sterilization is called Pasteurization. Boiling water, 100° 
C., kills vegetative bacteria in a very short time, less than two min- 
utes for most bacteria, and the spores of many species are destroyed 
by boiling for 5 to 30 minutes. 
Some spores, however, for example 
those of some varieties of B. vul- 
gatus, may survive a boiling tem- 
perature for several hours. Boil- 
ing is one of the most useful 
practical methods of disinfection. 
Nearly all pathogenic bacteria 
are quickly killed in boiling 
water. Surgical instruments are 
commonly boiled in water to which 
sodium carbonate, 1 to 2 per cent, 
has been added. Rusting and 
corrosion may also be prevented 
by adding 1o per cent of borax 
to the water in which metal in- 
struments are boiled. Steriliza- 
tion of bacteriological media is 
usually done by means of stream- 
ing steam, rather than by immer- 
sion in boiling water. The Koch steam sterilizer is a compara-, 
tively simple device for this kind of sterilization. It is a tall, 
cylindrical, tin vessel covered with asbestos or felt. The lower 
portion is filled with water; on the side is a water-gauge indicating 
the height of the water, in order that one may observe when there 
is danger of the sterilizer boiling dry. Over the top there is a 
tight-fitting cover. The steam is generated by a Bunsen burner 
standing underneath. A perforated shelf placed some distance 


Fic. 29.—Koch’s steam sterilizer. 


STERILIZATION—ANTISEPSIS—-FOOD PRESERVATION 67 


above the surface of the water is for the reception of the tubes 
and flasks that are to be sterilized. The Arnold steam steril- 
izer is somewhat more complicated but is very convenient and 
efficient. It consists of a cylinder of tin or copper with a cover, 
which is enclosed in a movable cylindrical outer cover or hood. 
The inner cylinder has an opening in the bottom through which 
steam may enter, the steam coming from a small chamber under- 
neath with a copper bottom to 


which the flame is applied. The 
peculiarity of this form of steril- 
izer consists in the fact that the = {{>-———————— 


steam which escapes from the 


sterilizing chamber condenses be- Pee ae ee 

neath the outer cover or hood 

and falls back upon the pan over |, Sterilizing Chamber a} 
the chamber in which thesteamis  §& H 


generated. The bottom of this 
pan is perforated with three small 
holes, which allow the water of con- 
densation to return into the cham- 
ber where the steam is generated. 
The sterilizer, therefore, to a cer- 
tain extent, supplies itself with 
water, although not by any means 
perfectly. It is, however, less 
likely to boil dry than other forms 
of sterilizers, and it has the ad- 
vantage of being reasonably cheap and quite effective. The 
space inclosed by the hood also serves as a steam-jacket and 
helps to prevent fluctuations in temperature. A great im- 
provement upon the ordinary Arnold sterilizer is the modifica- 
tion of it devised by the Massachusetts Board of Health. 

In the use of this, or any form of steam sterilizer, the time is 
noted from the period when boiling is brisk and it is evident that 
the sterilizing chamber is filled with hot steam; or, what is better, 


Fic. 30.—Diagram of the Arnold 
steam sterilizer. 


68 BACTERIOLOGY 


when the thermometer registers 100° C., if the sterilizer be pro- 
vided with a thermometer. With a large Arnold sterilizer a 
temperature of 100° C. may not be reached until it has been heated 
with a rose-burner for twenty to thirty-five minutes. When 
bulky articles or large amounts of material are to be sterilized, 
allowance must be made for the time necessary to bring the 
temperature in the middle of the mass to 100° C. 


Fic. 31.—Steam sterilizer, Massachusetts Board of Health. 


Autoclave Sterilization —Sterilization in the presence of moist- 
ure and at temperature above 100” C., requires a pressure greater 
than that of the atmosphere and the apparatus used for this pur- 
pose is known as the autoclave.- All bacteria and their spores are 
killed by heating at 110° C., in the presence of water, for fifteen 
minutes, and in about five minutes at 120° C. The steam pres- 
sures corresponding to these temperatures are approximately 
7+2 pounds and 15 pounds per square inch or 14 kilogram and 
1 kilogram per square centimeter, respectively. The autoclave 


STERILIZATION——ANTISEPSIS—-FOOD PRESERVATION 69 


consists of a metal cylinder with a movable top, which is fastened 
down tightly during sterilization. It is furnished with a pressure 
gauge, a stop-cock, and a safety-valve which is set to allow the 
steam to escape when the desired pressure is attained and thus 
prevents it from running too high. Heat is furnished by a gas- 
burner underneath. The lower part of the cylinder contains water. 
The objects to be sterilized are supported 
above this water on a perforated bottom 
or shelf. ‘ 

It is necessary to observe certain pre- 
cautions in the use of the autoclave. 
Allusion has already been made to the 
necessity for having the steam saturated 
with moisture. This is effected by allow- 
ing the air to escape after the heat is 
applied, and in order to be sure that all 
the air has really been expelled, the stop- 
cock, with which all autoclaves are pro- 
vided, is left open until the steam escapes 
freely. The stop-cock is then closed, and 
the pressure begins to rise. After leaving 
the articles to be sterilized in the auto- 
clave for the length of time desired, the JJ 
apparatus must not be opened while the 
steam contained within it is still under 
pressure, as there may be a sudden evolution of steam upon 
the removal of the pressure which may blow the media out of 


Fic. 32.—Autoclave. 


‘their tubes and flasks. After the pressure has fallen to zero it is 


well to open the stop-cock only a little way so that air may not 
be drawn in too rapidly to replace the condensing steam. The 
autoclave may be opened as soon as the internal and external 
pressure become equal. 

The length of exposure necessary to accomplish sterilization 
in the autoclave depends upon the protection which the article 
to be sterilized affords the bacteria. In sterilizing agar, a con- 


7° BACTERIOLOGY 


siderable interval elapses before the agar becomes liquified, es- 
pecially if it be in large flasks, and it is well to allow 30 to 35 
minutes at 110° C., for its sterilization. Closely packed surgical 
dressings serve to protect the interior, and considerable time may 
be required for penetration of a sterilizing temperature into such 
packages. In such instances it is unwise to rely upon the gauge 
as an indicator of the temperature throughout the materials 
being sterilized. It is well to test the efficiency of the steriliza- 
tion from time to time by enclosing test objects in the center of 
several packages. A convenient test object for surgical auto- 
claves may be made by spreading spores of B. subtilis or B. vulga- 
tus on a Sterile cover-glass and placing it in a sterile test-tube 
plugged with cotton, and then drying the preparation thor- 
oughly in the incubator for 24 hours. A number of these may be 
prepared and subsequently kept in the refrigerator until used. 
After the test object has been exposed in the autoclave, sterile 
broth is added to the tube by means of a capillary pipette. The 
development of culture from the spores indicates lack of effi- 
ciency in the process of sterilization. 

Discontinuous or fractional sterilization by moist heat is em- 
ployed to sterilize certain kinds of culture media, more especially 
blood serum and gelatin, which are likely to be injured by heat- 
ing above 100° C., or by prolonged heating. In this method the 
medium is exposed to a temperature deemed sufficient to kill the 
vegetative forms of bacteria but not the spores. An interval is 
then allowed for the generation of these spores, whereupon the 
cheat is again applied. This sequence is repeated until, according 
to past experience, sterilization may be regarded as almost cer- 
tainly accomplished. In the case of gelatin, steaming (100° C.) 
for 15 to 20 minutes on three consecutive days is the usual 
practice; with inspissated serum, exposure for 1 hour at 60° to 
70° C. on six successive days is usually sufficient. These methods 
are applicable only to media in which spores may germinate and 
they may fail to sterilize even in case of such materials, especially 
in the presence of rapidly growing spore-producing bacteria 


STERILIZATION—ANTISEPSIS—FOOD PRESERVATION 71 


and when there are spores of anaerobic bacteria in the material 
to be sterilized. On this account, materials sterilized in this way 
should not be injected into patients. 

Electricity has little or no direct demonstrable germicidal ac- 
tion. An electric current may generate sufficient heat to kill 
bacteria, pr it may produce powerful germicides by electrolysis, 
such for example as acids and alkalies. 

Chemical Agents.—Sterilization by means of chemicals is 
not employed in the preparation of culture media because of the 
difficulty of removing the added substance after the desired effect 
has been obtained. It is necessary in every case to consider 
the other effects which the use of chemical germicides entails, and 
their usefulness is therefore somewhat more limited than that of 
the physical agents for sterilization. Their efficiency is also 
subject to great variation according to the nature of the materials 
with which they come in contact. Nevertheless they have a 
very important place in practical sterilization and disinfection. 

The common soaps, and more particularly green soap, have 
a slight germicidal value, and this in conjunction with their sol- 
vent action upon fats and protein, and the mechanical cleans- 
ing which accompanies their use, justifies assigning them an 
important place among the chemical disinfectants. 

Acids, especially those which are strongly dissociated, are 
powerful germicides. Hydrochloric acid apparently owes its 
power entirely to its acidity, and in fairly weak solution, 0.2 to 
1.0 per cent, it kills vegetative bacteria in a short time. Strong 
sulphuric acid actually carbonizes organic matter, while nitric 
acid oxidizes and also forms special combinations with protein, 
the reactions resulting in death of living protoplasm. Sulphurous 
acid (sulphur dioxide) also possesses marked germicidal proper- 
ties, probably due to oxidation effects. 

Sulphur dioxide gas has been employed extensively in the 
fumigation of rooms, and is usually prepared by burning sulpbur. 
Much difference of opinion exists regarding the value of it as a 
disinfectant. The spores of anthrax .are not killed by several 


72 BACTERIOLOGY 


days’ exposure to the liquefied gas. Anthrax and other vegetative 
bacilli are destroyed in thirty minutes when exposed on moist 
threads in an atmosphere containing one volume per centum of the 
gas. An exposure of twenty-four hours in an atmosphere con- 
taining four volumes per centum of the gas will destroy the organ- 
isms of typhoid fever, diptheria, cholera and tuberculosis. The 
presence of moisture greatly enhances the activity of the disin- 
fectant, owing to the formation of the more energetic sulphurous 
acid. 

For the destruction of insects, such as mosquitoes, this agent 
is superior to formaldehyde. Its application for this purpose is 
important in preventing the spread of yellow fever and malaria. 

In practice, at least 3 pounds of sulphur per 1000 cubic feet 
should be used, and moisture must be present. This latter re- 
quirement can be fulfilled by evaporating several quarts of water 
within the tightly closed room just prior to generating the gas. 
In using powdered or flowers of sulphur, the necessary amount 
is placed on a bed of sand or ashes in an iron pot, which should 
rest on a couple of bricks in a pan or other vessel containing an 
inch or two of water. The sulphur is ignited by means of some 
glowing coals, or by moistening with alcohol and applying a 
match. Difficulty is often experienced in keeping the sulphur 
burning, and for this reason it is surer and more convenient to 
use the so-called sulphur candles. In operating with these, a 
sufficient number are placed on bricks in a pan of water and the 
wicks lighted. Liquefied sulphur dioxide may be used, and can 
be obtained in convenient tin receptacles containing a sufficient 
quantity for the disinfection of an ordinary room. The can is 
opened by cutting through a soft metal tube projecting from the 
top. The fluid vaporizes at the room temperature, and it is 
simply necessary to place the can in a convenient porcelain dish 
and allow the fluid to evaporate. © 

Sulphur dioxide is objectionable on account of its lack of 
germicidal power when dry, and on account of its corrosive action 
on metal and its bleaching effect on hangings and draperies in the 


STERILIZATION—ANTISEPSIS—FOOD PRESERVATION 73 


presence of moisture; it is, therefore, preferable to use formal- 
dehyde for room disinfection when possible. 

Alkalies, especially the caustics, sodium hydroxide and potas- 
sium hydroxide, are powerful germicides. Commercial lye is also 
valuable as a disinfectant. Perhaps the most important of the 
alkalies is calcium hydroxide, Ca(OH): which, because of its 
low cost, is extensively used for the disinfection of excreta. 

Lime.—The addition of 0.1 per cent of unslaked lime to fluid 
cultures of the typhoid bacillus and cholera spirillum will render 
them sterile in four or five hours. Typhoid dejecta are sterilized 
in six hours when thoroughly mixed with 3 per cent of slaked lime; 
the addition of 6 per cent will accomplish the same result in two 
hours. A convenient form for practical use is an aqueous mix- 
ture containing 20 per cent of lime—so-called milk of lime. 
Typhoid and cholera dejecta are sterilized in one hour after mix- 
ing with 20 per cent of this mixture. In practice it is safer to use 
a considerable excess of lime. From the foregoing facts it would 
seem probable that lime or whitewash as ordinarily applied would ~ 
possess disinfectant properties. Experimental work has demon- 
strated this to be a fact. The organisms of anthrax, glanders and 
the pus cocci are destroyed within twenty-four hours by one 
application. For spore-forming organisms and the bacillus of 
tuberculosis the power is not so great, the latter organism not 
being destroyed by three applications of the whitewash. Practi- 
cally, whitewashing is an effective means of disinfecting wood- 
work, perhaps because those microbes which are not killed at once 
are caught in the whitewash and their further distribution 
prevented. 

Oxidizing agents are usually germicidal. Chlorine, bromine 
and iodine, ozone, nitric acid, potassium permanganate, chlorin- 
ated lime, organic peroxides and peracids, and hydrogen peroxide, 
belong to this class. Chlorine, employed as chlorinated lime, 
is a valuable disinfectant for excreta. In the form of bleaching 
powder it has been extensively used in the disinfection of drinking” 
water and of swimming pools. Liquid chlorine is also employed 


74 BACTERIOLOGY 


for the same purpose. Bromine and iodine have long been 
employed in surgery, and solutions of iodine are often applied 
to the skin before surgical incision. Iodine probably acts to some 
extent as a germicide in this instance, but also as an antiseptic, 
remaining in the skin for some time after its application. Hydro- 
gen peroxide is a germicide, as it quickly decomposes to form 
water and oxygen. It is placed on the market in solutions 
varying in strength from 10 to 30 volumes, the mode of expression 
indicating that corresponding solutions will liberate ten to thirty 
times their volume of oxygen when appropriately treated. It 
decomposes rapidly when in contact with purulent secretions, 
setting free abundant oxygen, and on this account is used for cleans- 
ing infected wounds. It deteriorates in. strength so rapidly that 
only fresh solutions of known strength should be used. 

Potassium Permanganate—Koch asserts that a 3 per cent 
solution will destroy anthrax spores in twenty-four hours, but 
that a 1 per cent solution cannot be depended upon to kill patho- 
genic organisms. Its disinfectant value in practice is very low 
on account of its ready decomposition by inert material. In the 
dilute solutions usually used for medicinal injections and irriga- 
tions no disinfectant action occurs. 

Iodoform.—This substance possesses little if any disinfectant 
power. It is mildly antiseptic in moist wounds, due to the gradual 
liberation of small quantities of iodine. 

Inorganic Salts-—Mercuric chloride, HgCl:, is probably more 
commonly used than any other one germicide. But Geppert, 
whose work in this direction has been abundantly corroborated 
by others, found that the potency of corrosive sublimate as a 
germicide had been greatly overrated. The inhibitory action of 
corrosive sublimate, on the other hand, is very great, and the 
veriest trace of it left adhering to the bacteria is sufficient to 
prevent them from -growing. Corrosive sublimate is difficult 
to remove by ordinary washing and traces of it remain even after 
very thorough washing. But if the last traces are removed by 
treatment with ammonium sulphide or other reagents which pre- 


STERILIZATION—-ANTISEPSIS—FOOD PRESERVATION 75 


cipitate the mercury salt without themselves injuring the bac- 
teria, growth takes place even where the corrosive sublimate 
solutions have been used which are apparently efficacious. Thus 
anthrax spores will not grow in culture media when they are 
exposed for even a few minutes on silk threads to the action of 
corrosive sublimate solution of the strength of 149 per cent and 
then washed thoroughly in water and rinsed in alcohol; but 
Geppert showed that the spores so treated were only apparently 
killed, for it took twenty hours’ exposure to corrosive sublimate 
solution of this strength where the spores were not dried on silk 
threads, but suspended in water, and where the last trace of 
corrosive sublimate was removed by treatment with ammonium 
sulphide. It is claimed that its affinity for albuminous bodies 
and the readiness with which it combines with such substances 
detract frcm its value for some purposes. On the other hand, 
many observers claim that the albuminous combinations formed 
under such circumstances are soluble in an excess of albuminous 
fluid, and that its value as a germicide is not affected thereby. 
To obviate this possible difficulty it is customary in practice to 
combine the bichloride of mercury with some substance that will 
prevent the precipitation of the mercury salt by albumin. . For 
this purpose 5 parts of any one of the following substances to 1 
part of bichloride of mercury may be used—hydrochloric acid, 
tartaric acid, sodium chloride, potassium chloride, or ammonium 
chloride. A very practical stock solution for laboratory purposes 
has the following composition: 


Hydrochloric acid..............00-0005 100 C.C. 
Bichloride of mercury................-- 20 grams. 
Five c.c. in a liter of water makes a solution of about 1-1000 strength. 


Mercuric Iodide—An extremely high antiseptic value has 
been placed on this substance by Miquel, who claims that the 
most resistant spores are prevented from developing in a culture 
medium containing 1-40,000. In combination, as potassio-mer- 
curic iodide, it has been used in soaps (McClintock) with very 


76 BACTERIOLOGY 


‘favorable results. The substance is not extensively employed 
and further investigation. is necessary to dermine its true 
value. 

Silver Nitrate-—This salt probably occupies the position next 
to the bichloride of mercury in germicidal power. Behring claims 
it to be superior to bichloride of mercury in albuminous fluids. 
The anthrax bacillus is killed by a solution of 1-20,000 after two 
hours’ exposure. At least forty-eight hours’ exposure to a 1~10,- 
ooo solution is required to kill the spores of anthrax. It is very 
irritating, and possesses strong affinities for chlorides, forming 
with them insoluble chloride of silver, a salt without germicidal 
value. For these reasons the use of silver nitrate is limited. In the 
solutions usually employed for douching the cavities of the body- 
the available silver nitrate is immediately converted into the 
insoluble chloride, and little if any germicidal action takes place. 
To this fact may be ascribed the varying clinical results reported; 

Many proprietary silver compounds are on the market, in- 
troduced to replace the nitrate and its objectionable features. 
The most important are protargol and argyrol, organic silver 
combinations. They do not combine with chlorides, are less 
irritating than the nitrate and, not coagulating albumin, they 

“possess greater penetrating power. 

Organic Poisons.—Carbolic acid is one of the most important 
and most widely used disinfectants. It is usually employed in 
strengths of from 1 to 5 per cent. A 3 per cent solution will 
sometimes kill the spores of anthrax after two days’ exposure. 
In the absence of spores, the anthrax bacillus is destroyed by a1 
per cent solution in one hour. The less resistant pus cocci are 
destroyed rapidly by a 2 per cent solution. Combination with an 
equal proportion of hydrochloric acid enhances the efficacy of 
carbolic acid to a marked extent. This is due to the prevention 
of albuminous combinations, thus allowing greater penetration 
of the disinfectant. 

Many other substances closely related to carbolic acid are 
used and possess marked germicidal properties. Among them 


STERILIZATON—ANTISEPSIS--FOOD PRESERVATION 77 


may be mentioned creolin, cresol and lysol. They are all slightly 
superior to carbolic acid in actual germicidal value. 

Formalin is a 40 per cent aqueous solution of formaldehyde, 
H.CO. Numerous investigations have shown it to possess, both 
in the liquid and gaseous forms, remarkable disinfecting power 
under certain conditions. In solutions of 1-1000 an exposure of 
twenty-four hours is necessary to destroy the staphylococcus 
pyogenes aureus, while 1-5000 is sufficient to restrain its growth 
(Slater and Rideal). Its use in a gaseous form as a house 
disinfectant is by far the most important application at the present 
time. 

From 250 to 500 c.c. of formalin together with 500 to 1000 c.c. 
of water should be vaporized for each 1000 cubic feet of air space 
in the room, and the room should remain tightly closed for at 
least four hours, preferably over night. Many methods of 
vaporizing formaldehyde have been devised. Some form of 
tank, provided with heating apparatus and with an outlet tube 
which passes through the keyhole into the room, is perhaps the 
most convenient, where much disinfection has to be done. If 
apparatus of this sort is not at hand, good results may be obtained 
by putting the formalin and the water previously heated to boil- 
ing, in a large pail in the center of the room, and then adding 
rapidly crystalline potassium permanganate, about 200 grams to 
each 500 c.c. of formalin used. The permanganate oxidizes 
some of the formaldehyde and produces heat to evaporate the 
rest of it. From 25 to 50 per cent more formalin should therefore 
be used for a given air space. It is well also to add about 10 per 
cent of glycerin to the water so as to raise the boiling-point some- 
what and insure more complete vaporization of the formaldehyde. 

Formaldehyde penetrates very slightly beneath exposed 
surfaces so that everything to be disinfected should be completely 
exposed. Openings about windows and doors should be carefully 
plugged up and sealed with strips of paper. Mechanical cleansing 
supplemented by application of 11000 solution of mercuric 
chloride to floors and walls should follow the fumigation. The 


78 BACTERIOLOGY 


persistent odor of formalin may be removed by fumes of 
ammonia. 

Aniline Dyes——Many of the aniline dyes, notably pyoktanin 
(methyl-violet), possess germicidal properties. Malachite green 
is said to possess even greater germicidal value than pyoktanin. 
Methylene blue also possesses considerable germicidal power. 

Alcohol is a germicide of moderate power. It has little 
effect upon spores but in concentrations of from 50 to 95 per cent 
it destroys vegetative bacteria in a few minutes. 

Germicides destroy bacteria, as a general rule, because they 
are general protoplasmic poisons, destructive to all living matter. 
There is, nevertheless, some selective action. Thus, formal- 
dehyde kills bacteria but has little poisonous effect upon insects, 
such as mosquitoes, bedbugs, roaches or fleas. Mercuric chloride 
is rapidly fatal to bacteria when it comes into contact with them, 
but it has no very immediate destructive effect upon fly larve 
(maggots). Some of the oxidizing agents, such as hydrogen 
peroxide and acetozone are not poisonous to man because they 
are decomposed into relatively harmless substances before they 
can be absorbed. Attempts to discover or to produce chemicals 
which would exhibit a selective destructive effect upon microbes 
in the interior of the body have not met with much success. 
Quinine is perhaps the best known example, as it may circulate 
in the blood in sufficient concentration to poison the malarial 
parasites without at the same time killing the host. The effects 
produced by mercury and by salvarsan in syphilis are perhaps 
analogous, but they evidently depend to a large extent upon a 
special susceptibility of the microbe, a susceptibility not yet 
apparent in most parasites. The specific immune substances 
may perhaps be classed in the same category. These will be 
considered in more detail in a later chapter. 

Antiseptics.—Antiseptic and preservative agents prevent or 
delay the development of bacteria, without killing them. Very 
much the same agents are applied to prevent the growth of mi- 
crobes in living tissues and consequent poisoning of the body 


STERILIZATION—ANTISEPSIS—FOOD PRESERVATION 79 


(antisepsis) as in preventing microbic development in dead 
organic matter (food preservation). 

Of the physical antiseptics, desiccation and cold are perhaps 
of greatest importance. These agencies find application to the 
living body as well as in preservation of dead material. Sub- 
stances which increase osmotic pressure’ sodium chloride and 
sugar, are also employed to prevent microbic growth in foods. 

The chemical antiseptics are very numerous. In general 
a germicide in higher dilution exhibits antiseptic effect. “Small 
quantities of the inorganic acids, hydrochloric, nitric, sulphuric 
or sulphurous acid, prevent bacterial growth. Even boric acid, 
which has little or no germicidal effect, will delay or inhibit 
microbic development. Many organic acids possess inhibitive 
properties toward bacterial action. Acetic and lactic acids 
probably act merely by virtue of their acidity. Benzoic and sali- 
cylic acids seem to be more antiseptic, probably by virtue of other 
structural features in their molecules. Other organic substances, 
such as phenol (carbolic acid) and formaldehyde in high dilu- 
tions prevent or delay bacterial growth, and weaker germicides 
such as alcohol, chloroform or ether, are fairly effective preserva- 
tives. Oxidizing agents often decompose too rapidly to be of 
much value as antiseptics. Iodine, however, is one member of 
this group having considerable antiseptic value. 

Of the inorganic salts, mercuric chloride is most important. 
Small quantities of this agent inhibit the multiplication of bac- 
teria. It is extensively employed in antiseptic treatment of 
wounds. The borates, nitrates and salicylates, the latter com- 
pounds of an organic acid, also inhibit bacterial action to some 
extent. 

In using these substances as antiseptic applications to wounds, 
the possible poisonous effects upon the body as a whole from 
absorption of the antiseptic must be kept in mind. Moreover, 
such substances ought not to be used as food preservatives with- 
out due regard to the changes they may induce in the food and 
the possible effects they may exert upon the consumer. 


80 BACTERIOLOGY 


TesTING ANTISEPTICS AND DISINFECTANTS 


The determination of the antiseptic value of a material is a 
comparatively simple matter. A virulent culture of the organ- 
ism used as a test is inoculated into sterile bouillon containing a 
known quantity of the antiseptic. The process is repeated with 
varying strengths of the material until the smallest quantity of 
it capable of preventing growth is determined. This dilution 
may be considered the antiseptic value of the material in question 
for the organism used, under the conditions of the test. 

Determination of the disinfectant power of a substance is a 
problem of much greater magnitude, and the method used must 
be altered more or less to suit the properties of the substance 
tested. It is obvious that some of the substance tested remains 
in contact with the organisms in the method given for determin- 
ing the antiseptic value, and that we do not know whether the 
bacteria are alive and merely inhibited in growth, or actually 
killed. 

The chemical composition of the medium in which the bac- 
teria are tested may have a marked influence upon the action of 
germicides. If components of the medium enter into chemical 
union with the germicide there may be an inert compound 
formed. There may also be formed dense, flocculent precipi- 
tates which envelop the bacteria and protect them from the action 
of the germicide. It is therefore apparent that the potency of a 
germicide may appear very different when acting upon the bac- 
teria in water or in physiological salt solution or on bacteria 
dried on glass rods or on silk threads, on the one hand, and upon 
the same bacteria in beef broth or in feces or in urine, on the other. 
For these reasons it is not always possible to draw conclusions 
from the results of laboratory experiments as to the value of a 
germicidal agent for practical disinfecting purposes. 

Method—To 15 c.c. of sterile water in a 60 c.c. Erlenmeyer 
flask add 2 c.c. of a virulent culture of the test-organism. Then 
add a solution of the substance under investigation in the pro- 


STERILIZATION—ANTISEPSIS—FOOD PRESERVATION 81 


portion necessary to give the dilution wished. Mix thoroughly, 
and allow this ‘“‘action-flask”’ to stand as long as it is desired to 
have the germicide in contact with the test-organism (action- 
period). Transfer o.5 c.c. from the action-flask to a flask contain- 
ing 200 c.c. of a solution of some chemical capable of decomposing 
the substance being tested with the formation of inert or insoluble 
compounds. In this “inhibition-flask” the strength of the 
solution should be such that molecular proportions of the chemical 
are present in sufficient quantity to combine with all the germicide 
carried over. The inhibition-flask is shaken for 30 seconds, and 
1 c.c. transferred from it to 100 c.c. of sterile water in another, the 
“‘dilution-flask.” After two minutes, three agar tubes are 
inoculated with 1 c.c. each from the dilution-flask, plated, and 
growth watched for. 

Control-experiments should be performed to determine that 
the dilution of the test-culture is not too great when carried 
through the three flasks. It likewise should be determined that 
the inhibiting chemical has no effect on the bacteria. 

What the inhibiting chemical shall be must be determined 
for each individual case. For salts of the heavy metals ammo- 
nium sulphide answers well; for mercury salts, stannous chloride 
may be used; for formaldehyde, ammonium hydrate; for car- 
bolic acid, sodium sulphate. 

The testing of gaseous disinfectants, such as sulphur dioxide 
and formaldehyde, must be conducted under conditions as nearly 
parallel to actual practice as possible. The test-organisms may 
be exposed on threads or cover-glasses, and acted upon by a known 
volume strength of disinfectant for a known length of time. 
Subsequent treatment of the organisms with a suitable inhibitor 
is necessary when possible, and, should growth occur in the cul- 
tures following, the test-organism should be recognized in order 
that possible contamination by extraneous organisms may be 
excluded. . 

In determining the value of germicides for sterilizing ligatures, 


the students can apply methods based on the foregoing principles. 
6 


82 BACTERIOLOGY 


Great care and ingenuity are necessary to arrive at correct con- 
clusions, particularly in the case of animal tendons. In many 
instances quite stable compounds are formed between tendon 
and germicide, and living organisms may be so imbedded in such 
a substance that subsequent growth in a test-culture is impossible. 
The use of a suitable inhibitor, and, prior to final culture-tests, 
a prolonged soaking in sterile water, will promote the accuracy 
of the results. 


CHAPTER III 
CULTURE MEDIA 


Culture media are substances in which microbes are artificially 
cultivated. The variety of such substances is very large, different 
materials being suited to different purposes. Particular kinds 
of media have been devised in order to bring to development or 
especially to favor the development of certain kinds of microbes. 
Various media are also used to demonstrate the physiological 
properties cf bacteria, especially the physical arrangement of the 
bacterial cells as they grow under various conditions, and the 
chemical changes induced in the various constituents of the 
media by the microbic growth. 

Glassware.—Micro-organisms are usually grown in glass 
test-tubes or sometimes in glass flasks. The tubes and flasks 
should be of more durable glass than those ordinarily used in 
chemical work, but heavy tubes of glass of poor quality are not 
to be recommended. For ordinary purposes, test-tubes 125 X 
15 mm. are convenient. Larger tubes, 150 X 20 mm., are used 
to store media to be used in making plate cultures and for roll- 
tube cultures. New glassware should be thoroughly washed 
before using, and for critical work it should be boiled in dilute 
sodium carbonate, rinsed, washed in dilute hydrochloric acid, 
rinsed repeatedly in running water, finally in distilled water, 
and then inverted to drain in a warm place, such as the incubator, 
until perfectly dry. Used glassware should be sterilized in the 
autoclave at 120° C. for half an hour, emptied, cleaned with a 
swab and hot water, rinsed in distilled water and drained. In 
case of special difficulty the glassware may, after emptying and 
' washing in water, be cleaned by soaking in a special cleaning fluid, 


and all organic matter may be readily. removed by using this 
83 


84 BACTERIOLOGY 


fluid hot. It should not come into contact with the hands or 
with any large quantity of organic matter. 


t 


“CLeaninc FLum 


Water: pcwmmc nny oo xe ReX eae Y Oumar marnes 150 C.c. 
Dissolve the bichromate in water, with heat; 
allow it to cool; then add, carefully, con- 
centrated commercial sulphuric acid......... 230 C.C. 


Exact proportions are not necessary in making this fluid. Glass- 
ware cleaned in it must be repeatedly rinsed subsequently. 

Plugs.—The clean dry tubes or flasks are plugged with raw 
cotton of a good grade which does not char too readily upon- 
heating. The cotton plugs may be carefully made by rolling 
an oblong rectangular strip, of even thickness, into a firm cylinder 
of proper size, rolled plugs, or more hastily made by stuffing the 
cotton into the open end of the flask or tube, stuffed plugs. The 
latter kind of plug serves very well for tubes in which media are 
to be stored temporarily but is not so satisfactory for other 
purposes. 

Sterilization.—After plugging, the tubes are placed in a wire 
basket and sterilized in the hot-air sterilizer or, sometimes, to 
avoid charring, in the autoclave. This not only renders the 
glassware free from bacteria but also gives more permanent form 
to the plugs. 


THE CoMMoN CULTURE MEDIA 


Broth.—Broth, bouillon or beef-tea, is best made from fresh 
meat, either beef, veal or chicken. Finely chopped lean meat, 450 
to 500 grams, is mixed with 1000 c.c. of distilled water and either 
allowed to stand over night in the refrigerator or else digested 
for half an hour at temperature of 50 to 55° C. It is then strained 
through muslin, yielding a filtrate of deep red color. Any ex- 
cessive amount of fat should be skimmed off. To the filtrate, 
which should measure 1000 c.c., are added: 


CULTURE MEDIA 85 


Peptone?s «items gasvactacsw vignca eatpindcin duce bh Sel ro grams. 
Sodium chloride (common salt)................. 5 grams. 


These should be dissolved by stirring at a temperature below 
60° C. The mixture is then boiled for half an hour over the 
direct flame, cooled slightly, and filtered through paper pre- 
viously wet with warm water. ‘The filtrate should be clear and 
light yellow in color, and should be diluted to 1000 c.c. with 
distilled water. Its reaction is acid, a reaction unfavorable to 
the growth of many bacteria, especially to many pathogenic 
forms. 

The amount of alkali to be added is ascertained by titration. 
For this purpose exactly 5 c.c. of the broth is placed in each of 
three test-tubes. Five-tenths cubic centimeters of a 5 per cent 
solution of purified litmus (Merck’s highest purity) is added to 


each tube. An accurately prepared ee solution of sodium hydrox- 


ide? is then run in drop by drop from a graduated burette, the 
reading of which has been recorded, into one of the tubes until the 
red color. just changes to blue. The burette reading is taken 
and recorded. The alkali is then run into the second tube rather ~ 
rapidly until the endpoint ascertained by the first test is nearly 
reached. By comparing the color of this tube with that of the 
first one and with the third to which no alkali has yet been added, 
the exact point at which the color is changing from red to blue may 
be accurately judged. When this point is reached, the burette 
reading is again recorded and the amount of Alkali necessary 
to neutralize the 5 c.c. of broth ascertained. The third tube 
should then be titrated to confirm the previous result. The 
titration of the broth should now be repeated, using phenolphtha- 
lein as an indicator. For this purpose, 5 c.c. of the medium is 
transferred to a small porcelain dish, diluted by the addition 


1 Commercial peptones are mixtures of albumoses and a small amount of peptone. 
2 A normal solution of sodium hydroxide contains one aed aed of anhy- 


N 
drous NaOH, or 40 grams, in a liter. The > solution contains = of this amount 


or 2 grams in a liter. 


86 BACTERIOLOGY 


of approximately 45 c.c. of distilled water, and boiled for a minute. 
1 c.c. of a 0.5 per cent solution of phenolphthalein in 50 per cent 


alcohol is now added and ae solution of sodium hydroxide run in 


from the burette until the color changes to a faint but distinct 
and permanent pink color. The burette reading is recorded 
and the amount of alkali*necessary to neutralize the 5 c.c. of 
medium in respect to phenolphthalein thus ascertained. This 
titration may well be repeated, especially by beginners. As a 
result of these titrations we shall have ascertained’ the amount of 
alkali necessary to neutralize the remaining broth to either indi- 
cator. For example suppose that 5 c.c. of the broth titrated as 
follows: 


N eT, = 
0.5 c.c. of — alkali with litmus as indicator. 


2.0 C.c. of x alkali with phenolphthalein as indicator 


In order to. neutralize the remaining 980 c.c. of broth to litmus 


980 X 0.5 


_would require or 98 c.c oft alkali. A solution of 


alkali twenty times, as strong as this, namely normal sodium 
as as , 8 
hydroxide, is employed for this purpose, and only a or 4.9 C.c. 


of this are necessary to neutralize the 980 c.c. of broth to litmus. 
The reaction generally required for pathogenic bacteria is slightly 
alkaline to litmus and for this reason an excess of 10 c.c. of normal 
alkali per liter is added to the broth, 9.8 c.c. for the 980 c.c., mak- 
ing altogether 14.7 c.c. to be added. Calculation from the result 
obtained with phenolphthalein in the same way shows that 19.6 
c.c. of normal alkali would be required to neutralize the medium 
to this indicator. The desired final reaction of the medium 
‘in respect to phenolphthalein is acid, usually that of 5 to 15 c.c. 
of normal acid per liter, or 0.5 to 1.5 per 100 ¢.c., or 0.5 to 1.5 per 
cent, as it is commonly expressed after Fuller.!_ In this instance, 


* Fuller. Journal of Amer. Public Health Assoc., 1905. 


6 


CULTURE MEDIA 87 


therefore, 5 to 15 c.c. per liter, or 4.9 to 14.7 c.c. less than the 109.6 
for the 980 c.c., would be added, namely 14.7 to 4.9 c.c., accord- 
ing to the purpose for which the broth is to be used. 

The amount of normal alkali finally decided upon is added to 
the broth, which is then weighed in its pan. It is then cooked 
by boiling over the direct flame for half an hour or by heating - 
in the autoclave at 110° C. for 15 to 20 minutes. It is now cooled 
to about 50° C., filtered through paper, filled into tubes and 
sterilized, either in the autoclave at 110° C. for 15 minutes or by 
fractional sterilization in streaming steam at 100° C. for 15 minutes 
on three consecutive days. 

Broth may be prepared from meat extract instead of meat. 
Meat extract 3 grams, peptone 1o grams and salt 5 grams are 
dissolved in 1000 c.c. of water, boiled, filtered and titrated against 


ee : ; 
= sodium hydroxide. _The subsequent steps are the same as in 


preparation of broth from fresh meat. 

Remarks upon Titration—The titration of bacteriological 
media made from meat or meat extract is an important step in 
their preparation. There is some confusion on this point because 
of the use of different indicators in ascertaining the reaction. 
The neutral point indicated! by litmus is very nearly the actual] 
neutral point in respect to acidity and alkalinity, and this point 
is not appreciably displaced in either direction by the addition 
of a neutral mixture of a feebly dissociated acid and its salts to 
the solution. The end reaction indicated by phenolphthalein 
when it turns pink is actually a point at which there is a slight 
excess of alkali. This is so nearly the actual neutral point in 
inorganic solutions, when electrolytic dissociation is marked, 
that the error is not appreciable. In solutions of organic sub- 
stances, especially when considerable amounts of feebly dissociated 
substances, such as are contained in peptone or gelatin, are present, 


1 Washburn, E. W., The significance of the term alkalinity in water analysis and 
the determination of alkalinity by means of indicators. Report Illinois Waterworks 


Association, 1911. 


88 BACTERIOLOGY 


this error becomes very appreciable. The discrepancy between 
the end point for litmus and for phenolphthalein will vary for 
different lots of media. Another source of error and misunder- 
standing arises from the fact that the. reaction of a medium 
changes somewhat after its neutralization, especially during 
sterilization, but also upon standing afterward at ordinary tem-, 
perature. This change is toward decreased alkalinity and in- 
creased acidity and its extent is not the same for different media, 
being most marked, perhaps, in those rich in glucose. Where 
particular importance is attached to the titre of a medium, it is 
well, therefore, to determine this upon a sample of the medium 
taken from the lot at the time it is used, rather than to quote 
figures obtained before sterilization. The optimum reaction for 
most microbes is very close to the neutral point for litmus and 
for most pathogenic bacteria slightly alkaline to this indicator. 
A clear understanding of electrolytic dissociation and the 
measurement of acidity in terms of hydrogen ion concentration, 
requires a knowledge of the elementary principles of physical 
chemistry, which the beginning student of bacteriology may not 
possess. Inasmuch as it is customary to use such terms as 
P,, = 7.0 to indicate that a medium possesses neutral reaction, 
P,, = 6.0 for a definite slight acidity, and P,, = 8.0 fora definite | 
slight alkalinity, the student should be acquainted with them. 
When a crystalline salt, such as NaCl, is mixed with a quan- 
tity of a solvent insufficient to dissolve all of it, the salt exists in 
three states, (1) undissolved crystalline residue, (2) undissociated 
salt in solution as NaCl and (3) electrolytically dissociated salt 
in solution as the dissociated electrically charged ions, Nat and 
Cl-. If all of the solid substance is dissolved then it exists in the 
states indicated as (2) and (3). The speed of chemical reaction 
into which such a solution will enter depends upon the concentra- 
tion of the ionized portion. In the same way an acid, such as 
HCl, in solution in water is more or less completely ionized into 
Ht and Cl and the reactivity or chemical strength of such a 
solution is measured by the concentration of the ions and its 


‘CULTURE MEDIA 89 


acidity by the concentration of the hydrogen ion. Hydrogen ion 
concentration, therefore, is the physical measure of acidity. 
Pure water is in part a solution of dissociated H+ and OH- 
in undissociated H2O. Roughly the actual amount of dissociated 
H* in a liter of water is 0.000,000, 1 gram and the dissociated OH- 


is 0,000,001, 7 gram, in each instance representing a concentration 
I 


10,000,000 
Sérensen and others after him have employed the symbol Py 
to signify the logarithm of the reciprocal of the hydrogen ion 
concentration, thus omitting the minus sign, so that in pure water 
P, = 7.0. In a watery solution the concentration of hydrogen 


ions X the concentration of hydroxyl ions = a constant which is 
I 


100,000,000,000,000 
concentration increases, hydroxyl ion concentration diminishes. 
Thus a Deci-normal NaOH has an H* concentration of approxi- 
mately Normal X10-™. This is expressed on Sérensen’s scale as 
P, = 13. Conversely Deci-normal HCl has an H* concentration 
of about Normal X1o-! or P, = 1. 

In the presence of feebly dissociated compounds, phosphates, 
salts of organic acids and especially proteins, a large fraction 
of the hydrogen ions, introduced by the addition of a mineral acid, 
are quickly combined in the undissociated state. The same fate 
is met by hydroxyl ions introduced. The relatively lesser degree 
of dissociation in such solutions therefore requires the addition 
of greater amounts of acid or of alkali in order to bring about a 
definite alteration in chemical reaction or measured alteration 
in hydrogen ion concentration. This property of such solutions is 
spoken of as a buffer effect. Obviously bacteriological media are 
such as to exhibit this property in a conspicuous manner and to a 
different degree, according to their composition. 

Clark and Lubs! recommend a series of indicators which change 
color at various hydrogen ion concentrations and their chart has 


Normal or Normal X 107’, which is Normal log — 7.0. 


Normal or Normal X1071*, As hydrogen ion 


* Clark, W. M. and Lubs, H. A.: Journ. Bact., 1917, li, £, 109, 191. 


go 


or 


w 
uolze1908S1q 


Fic. 33,—Dissociation curves of indicators considered as simple mono-basic acids, showing percentage of color change with 


BACTERIOLOGY 


(After Clark and Lubs.) 


Px. Shaded portions of curves indicate the useful ranges. 


been used by the Committee on 
the Descriptive Chart of the Society 
of American Bacteriologists. To the 
left of P, =7 is the acid range and 
to the right of 7 the alkaline range. 
The useful range of each indicator is 
shown by the heavily shaded portion 
of the respective curve. 
Gelatin.—Finely chopped meat, 
450 to 500 grams, is mixed with a 
liter of distilled water and. digested 
on the water bath for half an hour 
at 50-55°, with stirring. It is then 
strained through muslin, yielding a 
filtrate of deep red color, which 
should be made to equal] 1000 c.c. 
This filtrate is placed in the inner 
compartment of a double boiler (rice 
cooker) and to it are added 10 grams 
peptone, 5 grams sodium chloride 
and 100 to 150 grams of sheet gelatin 
of the best quality (“gold label” 
gelatin). The larger amount of 
gelatin should be used during warm 
weather if no low temperature in- 
cubator is at hand. These con- 
stituents are dissolved by stirring at 
a temperature below 55°C. After 
complete solution, the reaction is 
titrated as has been described for the 
titration of broth. From 30 to 50 
c.c. of normal alkali are usually re- 
quired to give the proper reaction 


1Conn, H. J. and others: Journ. Bact., 
1919, LV, 107. 


CULTURE MEDIA gi 


to a liter of the medium. After this has been ascertained, and 
the amount ‘added, the medium is thoroughly mixed and then 
left covered and undisturbed while the water in the outer com- 
partment of the cooker is boiled for an hour. It is well to have 
boiling water at hand in another receptacle so that the supply 
in the cooker may be replenished if it gets low, without chilling 
the medium. The gelatin is now filtered through paper wet with 
hot water, and should be kept warm during 
filtration by means of a funnel-heater, or by 
a steam bath, although these are not indis- 
pensable. If it gets cold it may be poured 
out of the funnel and warmed again in the 
pan. A portion of the filtrate should be 
boiled in a test tube over the flame for a 
minute or two. It should then remain 
(1) perfectly clear, (2) alkaline to litmus 
paper, and (3) should solidify on cooling in 
tap water. After filtration the medium is 
filled into tubes and sterilized in streaming 
steam by the fractional method, 20 minutes 
at 100°C. for 3 consecutive days. Gela- 
tin may be sterilized in the autoclave at 
110°C. for ro minutes, but it should be 5. reer rere ee 
chilled in cold water at once after removal, filling media into tubes.’ It 
, i bs Se isheld in a ring-stand sup- 
and even then its gelatinizing property port. 
may be seriously impaired. 

In filling gelatin into tubes it is important that the medium 
should not be spilled on the mouth of the tube cr on the cotton 
plug, as this accident causes the latter to be glued in position. 
The filling apparatus indicated in Fig. 34 will be found convenient 
for filling any sort of liquid medium into tubes, and with proper 
care one may fill tubes rapidly without soiling the mouths of tubes 
and their cotton plugs. 

Gelatin may be made from beef extract. The extract, peptone, 
salt and gelatin are dissolved at a temperature below 60°C. or 


92 BACTERIOLOGY 


the medium is cooled to this temperature after solution has been 
accomplished. It is titrated and the proper amount of alkali 
added. An egg is beaten up in water and then stirred into the 
medium. It is then boiled on the water bath for an hour, filtered, 
tested, tubed and sterilized. 

Nutrient Agar.—To a liter of nutrient broth, prepared as 
above described (page 84) add 15 grams of finely cut agar shreds. 
Weigh the pan with its contents. Boil the material over the 
direct flame for one to two hours, with constant stirring to avoid 
burning, adding hot distilled water from time to time to compensate 
for the loss by evaporation. Instead of boiling it is convenient 
to cook the medium in the autoclave at 110°C. for 45 minutes 
to an hour. In either case, the agar should be very completely 
dissclved. The medium is then cooled to 60°C. and an egg pre- 
viously beaten up in water is added and thoroughly mixed with the 
agar. It is then boiled again for 10 minutes over the free flame, 
with constant stirring at the bottom, or for 45 minutes on the 
water bath, or for 15 minutes in the autoclave at 110°C. Distilled 
water is added to restore the original weight, and the medium is 
then filtered, usually through a layer. of cotton wet with hot 
water, although filter paper may be used. Filtration is favored 
by keeping the funnel hot, cither with the hot-wate1 funnel: 
heater or in a steam bath, and it may be hastened by the usé of 
suction. The filtrate need not be perfectly clear, and it usually 
clouds on cooling unless it is acid in reaction. The reaction 
should be alkaline to litmus. After filling into tubes or flasks, 
agar should be sterilized in the autoclave at 110°C. for 30 to 
35 minutes. 

Time may be saved by using the more expensive powdered 
agar in place of the agar shreds and when a very clear medium 
is desired the liquid agar may be allowed to sediment in a water 
bath at 60° C. for some hours before filtration. 

Modifications of the Common Media.—Broth is made nearly 
free from sugar by fermenting the meat infusion over night at 
37°C. after inoculating it with B. coli, and then proceeding with 


CULTURE MEDIA 93 


the filtrate in the usual way. This medium is designated as 
sugar-fiee broth. Various sugars or other substances are added 
to such Eroth in order to test the ability of bacteria to ferment 
them. Acetic acid, 0.5 per cent, is added to broth to make a 
selective medium for acid-resisting bacteria. Glycerin, 5 to 7 
per cent, is added to broth for the cultivation of the tubercle 
bacillus. Naturally sterile ascitic fluid or blood is a, 
added to broth to promote the growth of certain types rs: 
of microbes, and to encourage anaérobes. Bits of | 
naturally sterile tissue are added to broth for similar 
purposes. 

Gelatin is modified by the addition of various sugars, 
especially dextrose and lactose, often with the further 
addition of litmus. The production of acid by fer- 
mentation of the sugar is at once evidenced by the 
reddening of the litmus. Glucose litmus gelatin is also 
a useful medium for anaérobes. It is best to sterilize 
the litmus separately and add it from a sterile pipette 
at the time the medium is used. 

Agar is modified by the addition of 5 to 7 per cent 
of glycerin, and such glycerin-agai is used extensively 
for cultivation of the tubercle bacillus and several 
other pathogenic bacteria. Various sugars, supple- 
mented by the addition of litmus, are dissolved in 


. fic. 45" 
agar to test the fermentation properties of bacteria. Potato in 


Glucose agar is extensively employed as such for the culture tube. 


cultivation of anaérobes. Agar also forms the gelatinizing base 
for a number of more or less complex special media. 


STERILIZABLE SPECIAL MEDIA 


Potato.—Potatces were perhaps the first solid medium em- 
ployed in the cultivation of micro-organisms. Boiled or steamed 
potatoes kept in a moist place, such as a large covered glass dish, 
may well be employed as an illustration of primitive technic, and 
excellent cultures of the common chromogenic bacteria may be 


94 BACTERIOLOGY 


obtained in this way. For most purposes it is better to put pieces 
of potato in test-tubes where they are more perfectly protected 
from contamination, as suggested by Bolton. The potato is 
carefully washed, a slice removed from each end, and a cylinder 
is cut out with a cork-borer or with a test tube cut off near its 
bottom. This cylinder is divided diagonally into two pieces. The 
pieces are washed in running water for twelve to eighteen hours. 
' They are placed in test-tubes containing a little water to keep 
the potato moist, and are supported from the bottom on a piece 
of glass tubing about 1 to 2 cm. in length (or in cotton, or in a 
specially devised form of tube with a constriction at the bottom). 
The tubes are plugged, and sterilized in the autoclave at 110° C. 
for 30 minutes. Potato is best when freshly prepared; it is likely 
to become dry and discolored with keeping. 

Milk.—Milk fresh as possible is placed in a covered jar, 
steamed for fifteen minutes, and then kept on ice for twenty-four 
hours. At the end of that time the middle portion is removed 
by means of a siphon. The upper and lower layers must not be 
taken; the upper part contains cream, and the lower part par- 
ticles of dirt, both of which are to be avoided. About 7 to 10 
c.c. are to be run into each test tube. The tube is plugged with 
cotton, and sterilized in the autoclave at 110° C. for 30 minutes. 

The coagulation of milk, which is accomplished by certain 
bacteria, is a very valuable differential point. A little litmus 
tincture may be added to the tubes of milk before sterilizing, 
until they acquire a blue color, to indicate whether or not acids 
are formed by the bacteria which are afterward cultivated in 
the milk. Better results are obtained by sterilizing the litmus 
solution separately and adding it to the sterilized milk with 
aseptic technic. 

Dunham’s Peptone Solution. 


PePtON moicag ues s sewsess eee a ys amtee ue eeeE fo grams. 
Sodtitiy Chlorid Guess waduws wanda ee aacgns cmscs oe 3 grams. 
WA LET: tea eicoin merck eo endeared ate al ear e Ran thse Ben 1 liter. 


Boil, filter, sterilize in the usual manner. 
' Bolton, The Medical News, Vol. 1, 1887, p. 318. 


‘ CULTURE MEDIA 95 


Dunham’s solution is valuable to test the development of 
indol_ by bacteria (see Part II., Chapter VIII.). The develop- 
ment of acids may be detected after the addition of 2 per cent of 
rosolic acid solution (0.5 per cent solution in alcohol); alkaline 
solutions give a clear rose-color which disappears in the presence 
of acids. _ 

Nitrate Broth.—Dissolve 1 gram of peptone in tooo c.c. of 
distilled water, and add 2 grams of nitrite-free potassium nitrate. 
Fill into test-tubes, ro c.c. in each, and sterilize in the autoclave 
at 110°C. for 15 minutes. 

Blood-serum.—The blood of the ox or cow may be obtained 
easily at the abattoir. It should be ‘collected in a clean jar. 
When it has coagulated, the clot should be separated from the 
sides of the jar with a glass rod. It may be left on the ice for 
from twenty-four to forty-eight hours. At the end of that time 
the serum will have separated from the clot and may be drawn 
off with a siphon into tubes. These tubes are sterilized for the 
first time in a slanting position, as the first sterilization coagulates 
the serum. The coagulation may be done advantageously, as 
advised by Councilman and Mallory, in the hot-air sterilizer at a 
temperature below the boiling-point. After coagulation, sterilize 
in the autoclave at 110° C. for 20 minutes. This serum makes an 
opaque medium of a cream color. Blood-serum may be more 
conveniently sterilized in the Koch serum inspissator (Fig. 36). 
A clear blood-serum is to be obtained by sterilization at a tempera- 
ture of 58° C. for one hour, on each of six days, if a fluid medium 
is desired, or of 75° C. on each of four days if the serum is to be 
solidified. In the latter case the tubes are to be placed in an in- 
clined position. Opaque, coagulated blood-serum has most of 
the advantages of the clear medium. Blood-serum may be se- 
cured from small animals by collecting blood directly from the 
vessels, and with proper technic may be obtained in a sterile 
condition; and the serum may be separated and stored in a fluid, 
state. Human blood-serum is sometimes obtained from the 
placental blood. The preservation of blood-serum is sometimes 


96 BACTERIOLOGY 


accomplished with chloroform, of which 1 per cent is to be added 
to the medium; in this manner the serum may be preserved for a 
long time. It may be filled into tubes, solidified and sterilized 
as required; the chloroform will be driven off by the heat, owing 
to its volatility. Blood-serum media which are sterilized at 
low temperatures should be tested for twenty-four hours in the 
incubator to prove that sterilization has been effective; if it has 
not, development of the contaminating bacteria will take place 
and be visible to the eye. 


Fic. 36.—Koch’s serum sterilizer. 


Léffler’s blood-serum consists of one part of bouillon con- 
taining 1 per cent of glucose, mixed with three parts of blood- 
serum. It is sterilized like ordinary blood-serum. It is used 
largely for the cultivation of the bacillus of diphtheria. 

Fresh eggs in their shells may be used without other prepara- 
tion than washing the surface thoroughly with bichloride of mer- 
cury solution; or after sterilization by steam, which of course 
coagulates the albumen. The egg is easily inoculated through a 
small opening made with a heated needle, which may be closed 
afterward with collodion. Hueppe recommended eggs closed in 
this manner for the cultivation of anaérobic bacteria. 


CULTURE MEDIA 97 


Dorset’s Egg Medium.'—Perfectly fresh eggs are washed and 
the shells sterilized with bichloride solution. The eggs are then 
carefully broken and the yolks and whites mixed in a sterile 
dish. The mixed material is poured into sterile tubes and solidi- 
fied in the slanting position by heating at 7o—75° C. for two 
hours. Contamination with bacteria should be carefully avoided 
throughout the preparation of the medium. The tubes should 
be sealed with rubber caps or with wax and incubated for a 
week before use. It is well to moisten the surface with a few 
drops of sterile water from a pipette before inoculating. This 
medium is used for growing the tubercle bacillus. 

Bread-paste.—Dry or toasted bread is broken into small 
crumbs, filled into tubes or flasks, moistened with water and 
sterilized in the autoclave. This medium is used for cultivation 
of molds. 


MEDIA CONTAINING UNCOOKED PROTEIN 


Culture media containing naturally sterile uncooked protein 
have made possible the cultivation of microbic forms not cultivable 
on other media. Many microbes which may also grow on cooked 
media do much better on those containing uncooked protein. It 
would seem that media of this kind are to play an important part 
in the further development of our knowledge of pathogenic 
micro-organisms. 

Collection of Sterile Blood.—A few drops of blood may be ob- 
tained from the ear lobe. The skin is cleansed with soap and 
alcohol and then dried perfectly with sterile ‘cotton. It is 
punctured with a sterilized lancet and the blood quickly trans- 
ferred to the surface of an agar slant by means of a platinum 
loop or a sterile capillary pipette. It should be incubated before 
use to insure sterility. 

Larger quantities of sterile human-blood may be obtained with 
far less danger of contamination from the median basilic vein or 
other large vein at the elbow. The skin is washed, disinfected 


1 Dorset: American Medicine, April 5, 1902. 
7 


98 BACTERIOLOGY 


with alcohol and bichloride and dried. An elastic bandage is 
applied about the arm to distend,the veins. A sterile neédle 
attached to a special sterilized blood pipette is thrust into the 
vein and the desired amount of 
blood collected (see Fig. 37). It 
may be allowed to clot if sterile 
serum is desired, or it may be 
defibrinated by stirring with the 
glass rod if a mixture of corpuscles 
and serum is desired, or it may be 
kept in the fluid state by the ad- 
dition of sterile 10 per cent solution 
of sodium citrate so that the final 
mixture may contain 1 per cent of 
citrate. The bandage is removed 
from the arm before the needle is 
withdrawn. Pressure over the 
wound with cotton wet in alcohol 
for five minutes prevents sub- 
cutaneous hemorrhage. No dress- 
ing is required. The inlet to the 
blood pipette is closed by kinking 
the rubber tube. The blood or the 
serum is subsequently handled by 
means of sterilized pipettes, and 
most conveniently by means of 


Fic. 37.—Pipette with needle at- the Pasteur bulb pipettes. (See 
tached for drawing human blood 
from a vein for use in culture media. pape 33-) 
The glass rod inside is used to defi- Blood from smal} laboratory 


Dabiaae Che Hipues animals serves as well as human 
blood for most purposes. It may be drawn from the carotid artery 
by aseptic technic into a special blood pipette, the lower end of 
which is drawn out into a capillary ,which is inserted directly into 
the artery (see Fig. 38). This blood may be defibrinated, ci- 
trated or allowed to clot. 


CULTURE MEDIA 99 


Small amounts of sterile blood may be obtained from labora- 
tory animals without killing them by means of heart puncture. 
The needle of a Luer glass syringe is inserted through the chest 
wall, after anesthetizing the animal and shaving and disinfecting 
the skin, so as to enter the cavity of the right ventricle. A 
quantity of blood not greater than 19 the weight of the animal 
may be removed. The needle is withdrawn 
and the blood quickly forced out into a sterile 
tube where it may be defibrinated or mixed 
with citrate solution, or allowed to clot, as 
may be desired. 

Very large amounts of sterile blood are 
best obtained from the juglar vein of the 
horse or the superficial abdominal veins of 
the cow. The skin is shaved, washed and 
cauterized with 95 per cent carbolic acid. 
When this has dried the vein is punctured 
with the needle, which is attached to a 
suitable glass receptacle by means of rubber 
tubing. 

Collection of Sterile Ascitic Fluid.— 
For this purpose a large trochar and canula 
provided with a lateral outlet, and made so 
that the trochar can be drawn back beyond 
this outlet without being completely removed, 
is most convenient. The instrument is oiled 
with liquid paraffin. A rubber tube about _ Fic. 38.—Pipette with 

a : ‘ capillary tip for drawing 
40 cm. in length is attached to the outlet  piood from carotid artery 
and the whole is wrapped in a cloth and re ean, age 
sterilized in the autoclave. The site selected 
for puncture should be cleansed and painted with tincture of iodine 
and the skin may be frozen with ethyl chloride if desired. One 
man inserts the trochar and canula, taking care not to contaminate 
it after it is removed from the cover. Another manipulates the 


attached rubber tube, carefully guarding it from contamination 


Ico BACTERIOLOGY 


and allowing the fluid to flow into sterilized flasks of 1000 c.c. 
capacity which are handled by an assistant. The mouth of each 
flask should be flamed after removing the cotton plug and again 
before it is inserted after filling the flask. With proper technic 
the ascitic fluid will as a rule be found bacteria-free. It should 
be stored in a cool place, and is most conveniently handled by 
means of large Pasteur bulb pipettes. 

In collecting hydrocele fluid or other fluids to be 
used for’ culture media, similar aseptic technic should be 
employed. ° 

Sterilization of Contaminated Fluids.—Any of the clear fluids 
may be sterilized, when this is necessary, by filtration through the 
Berkefeld filter. The filtrate will usually prove less valuable 
as a medium than the corresponding unfiltered naturally sterile 
material. 

Collection of Sterile Tissue.—For this purpose, a healthy 
animal is first bled to death as described above (page 98) for the 
collection of sterile blood. The skin is then thoroughly wet with 
water or with bichloride solution. With sterile instruments, an 
incision is made in the median line and the skin carefully stripped 
back. It is then well to sear the abdominal wall with a hot iron 
along the median line and also crosswise and cut along these 
lines with sterile scissors, opening the abdominal cavity. The 
organs desired are quickly removed with sterile instruments 
and placed in covered sterile glass dishes. ‘The liver, kidneys 
and testes are the organs most frequently employed in culture 
media. They are divided into pieces of suitable size with sterile 
scissors. Brain tissue may be readily obtained from the rabbit. 
The top of the head is skinned and an opening made by cutting 
away the skull between the orbits with the bone forceps. An area 
of the anterior portion of the brain is exposed. This is thoroughly 
seared with a hot iron, as well as the adjacent structures. A 
Pasteur bulb with a large capillary (internal diameter at least 5 
mm.) is convenient for drawing out the brain tissue. This large 
capillary is inserted through the seared area and the brain is 


CULTURE MEDIA : IOI 


broken up by moving it about in the cranial cavity, while the 
tissue is drawn into the bulb by suction. 

Pfeiffer’s Blood-streaked Agar.—A large loopful of naturally 
sterile human blood, freshly taken from the ear, is spread over 
the surface of an agar slant, and incubated to insure sterility. 
This medium is employed for cultivation of the influenza 
bacillus. 

Novy’s Blood-agar.—The agar is melted and cooled to 50° 
C. The naturally sterile defibrinated blood, usually rabbit’s 
blood, is warmed to about 40° C. The blood is mixed with the 
agar in various proportions, and the mixture is allowed to solidify 
in the inclined position. The medium should be fairly firm in 
consistency and some fluid should collect at the bottom of the 
slant. The medium is useful for cultivation of the gonococcus, 
the influenza bacillus, streptococcus, pneumococcus and meningo- 
coccus, but more especially for cultivation of the flagellated 
hematozoa such as trypanosomes and related organisms, including 
the various species of Leishmania. 

Smith’s Broth Containing Sterile Tissue.—Pieces of naturally 
sterile organs, usually liver or kidney, are placed in broth, more 
particularly in fermentation tubes of broth. The bits of tissue 
are conveniently handled by touching with a hot platinum wire 
or glass capillary, to which they will adhere. The medium is 
especially useful for the culture of anaérobic bacteria. Naturally 
sterile blood added to the broth also serves for this purpose. 

Ascitic-fluid-agar.—This is made in the same way as the 
Novy’s blood-agar except that naturally sterile human ascitic 
fluid is employed instead of blood. The medium is beautifully 
transparent, and may be employed for plating as well as for tube 
cultures. It is especially valuable for cultivation of the gonococcus 
and also for the streptococcus, pneumococcus and meningococcus. 

Noguchi’s! Ascitic Fluid with Sterile Tissue.—Naturally 
sterile tissue is placed in a tall tube. A deep layer of ascitic fluid 
is added, and for some purposes this is covered with a layer of 

1Noguchi: Journ. Exp. Med., Jan. 1, 1912, Vol. XV, pp. go-100. 


102 BACTERIOLOGY 


sterile paraffin oil. The medium is used more especially for 
the cultivation of the blood spirochetes which cause relapsing 
fever. : — 

Special Media of very special and limited usefulness will be 
discussed ih the chapters dealing with the particular organisms 
for the growth of which they are employed. 


CHAPTER IV 


COLLECTION OF MATERIAL FOR BACTERIOLOGICAL 
STUDY 


Bacteria under natural conditions are usually associated as 
mixtures of several species living together. Only under rather 
exceptional circumstances will a single kind of bacteria be found 
growing alone. This does occur in disease, however, where the 
living host may be able to keep out all but the one kind of microbe. 
But even diseased tissues or exudates originally harboring only 
one kind of bacteria may quickly acquire others in abundance 
after removal from the living body. It is well therefore to 
regard any material presented for bacteriological examination 
as potentially, and in all probability actually, harboring several 
kinds or species of bacteria. The direct planting of such material 
on a culture medium will, therefore, in most instances give rise 
to a mixed culture, in which those forms least prominent in the 
original material may easily appear as most important. If the 
material be allowed to stand, especially if it be a favorable medium 
for bacterial growth, the relationships present may become 
seriously confused. It should, therefore, be examined as fresh 
as possible. When immediate examination is impossible the 
material should be kept on ice. 

Samples of water, milk or other fluid should be collected in 
sterilized tubes or bottles. Samples of solid food should be 
seared or charred all over the surface and divided with a sterilized 
knife. A small piece of the interior is then removed to a sterilized 
glass dish and covered. 

Material removed from the human or from the animal body during 
life or at autopsy may be bacteria-free, or it may contain one or 
‘ more species of microbes. It is important that the picture 
be not confused by the addition of bacteria from the surface of 

103 


104 BACTERIOLOGY 


the body, from instruments or from the air during the collection 
and transportation to the laboratory. Unfortunately the lab- 
oratory study of such material is too often rendered difficult, un- 
trustworthy or even worthless through lack of attention to this 
point. 

When merely microscopic examination is to be undertaken, 
contamination may not be serious, and an antiseptic, such as 
two per cent of carbolic acid; may be added to the material, if 
fluid, and if solid it may be immersed in ten per cent formalin. 
The bottles used should be new and clean. Such material may 
also be spread on microscopic slides or cover-glasses in a thin- 
layer, dried, fixed in the flame, and transported to the laboratory. 
This method is not always free from danger when the material 
passes through several hands. Special precautions for collecting 
material for microscopic examination will be considered in discuss- 
ing the specific pathogenic microbes. 

Specimens of sputum should be raised from the trachea, bron- 
chi and lungs after previously cleansing the mouth. Sputum 
should be received into a sterile wide-mouthed bottle, and stop- 
pered with a sterilized:cork. The exterior of the bottle should 
then be carefully washed with 5 per cent carbolic acid. 

Urine should be collected by catheter with careful aseptic 
technic, and should be received in a clean sterilized bottle. When 
the passage of a catheter is deemed unwise, the urine should be 
received directly into a sterile bottle after surgical cleansing of- 
the urinary meatus and must be examined in the laboratory with-* 
out delay, contamination being assumed to have taken place. 

Blood and transudates are collected by the technic previously 
described (page 97). Blood is drawn from the vein by means 
of the Luer syringe and is quickly ejected into several flasks of 
broth (150 to 250 c.c.) and into Petri dishes where it is mixed with 
melted agar (cooled to 50° C.) before clotting takes place. 

A most convenient tube for collecting blood has been described 
by Taylor.’ The side arm (Fig. 39) is accurately ground at the 

‘Taylor, R. M.: Proc. N. Y. Path. Soc., 1914, 14, p. 37. 


. 


MATERIAL FOR BACTERIOLOGICAL STUDY 105 


tip to fit the ordinary Luer needle. The tube is sterilized by hot 
air and the needles are sterilized by boiling in liquid paraffin 
(albolene). By putting 1 c.c. of ro per cent sodium citrate into 
the tube, the blood specimen may be kept fluid and transported 
a considerable distance to the laboratory before inoculating 
the culture media. 


wy 


Fic. 39.—Taylor’s tube for vein punctures. 


Cerebro-spinal fluid is obtained by inserting a sterilized needle 
(4 cm. long for children, 8-10 cm. long for adults, and with lumen 
1 mm.) in the median line in the back, so that it enters the spinal 
canal between the second and third, or between the third and 
fourth, lumbar vertebra. Aseptic technic is essential. The fluid 
coming from the needle is received in a sterile tube. 

Feces from infants and young children are best collected by 
means of a heavy glass tube closed and rounded off at the end, and 
provided with a lateral opening near the closed end. This is 
enclosed in a larger tube and sterilized. It is inserted well into 
the rectum with aseptic technic and the entrance of fecal material 


106 BACTERIOLOGY 


through the lateral opening is favored by gently moving the tube. 
It is then withdrawn and replaced in its original container to be 
transported to the laboratory. From adults the feces are passed 
directly into a sterilized covered agateware basin without other 
special apparatus. 
; Intestinal juice from the duodenum may 
be obtained in infants? by inserting a sterile 
4 rubber catheter, closed below with asterilized 
i gelatin capsule, through the esophagus and 
fH stomach into the duodenum. The capsule 
His then blown off. by pressure from a sterile 
Hl syringe attached at the other end of the 
Hl catheter and the fluid contents of the duode- 
: num aspirated. In adults? the Einhorn duo- 
f denal tube is employed. The tube is steri- 
: lized by boiling and the lower opening sealed 
i] with a sterilized gelatin capsule and by 
H finally coating with shellac. The tube is in- 
w(@| serted through the esophagus and is carried 
) through the pylorus by peristalsis. Ordi- 
y narily it is inserted in the evening. On the 
instrument for obtaining following morning the seal at the lower end 
ee is broken by pressure of a sterile syringe 
ae Schmidtand attached to the free end of the tube and 
the sample of juice aspirated. Intestinal 
juice may be obtained from various levels in the jejunum also by 
regulating the length of tube inserted. 

Pus and other exudates are best collected in sterile glass capil- 
lary pipettes (see page 33). A sterilized cotton swab, made 
by winding a pledget of absorbent cotton around the end of a stiff 
wire, enclosing it in a test-tube and sterilizing it, is also useful, 
especially when it is impossible or undesirable to employ the 
glass tube. 


' Hess: Journ. Infectious Diseases, July, 1912, Vol. XI, pp. 71-76. 
* MacNeal and Chace: Arch. Int. Med., Aug., 1913, Vol. XII, pp. 178-197. 


MATERIAL FOR BACTERIOLOGICAL STUDY 107 


At autopsies on human subjects, the same principles for col- 
lection of material apply. Fluids are best collected in sterile 
glass pipettes and even solid organs may be seared and punctured 
with a strong glass capillary into which some of the pulp is drawn 
by suction. The tubes may be sealed in the flame and_ trans- 
ported considerable distances to the laboratory. This is usually 
more satisfactory than the inoculation of culture media in the 
autopsy room, especially if the facilities for bacteriological work 
there are somewhat limited. Smears on slides or cover-glasses 
should also be made for microscopic examination, and pieces of 


the various organs fixed in alcohol or formalin and preserved 
for sectioning. 


CHAPTER V 
THE CULTIVATION OF MICRO-ORGANISMS 


Avoidance of Contamination.— Micro-organisms are so numer- 
ous on the body of man and in his environment that they are likely 
to be present on all articles about us unless special precautions 
are taken to remove or destroy them. The dust blown about 
in the air contains bacteria and spores of molds. The primary 
essential in all bacteriological culture work is the exclusion of 
these extraneous micro-organisms. The unskilled or careless 
worker may quickly add some of these chance organisms to the 
material which he is attempting to study, introducing an element 
of almost hopeless confusion unless it is recognized. Ancther 
essential of great importance, especially when working with patho- 
genic microbes, is the complete destruction of all living bacteria 
before they are allowed to pass beyond strict and absolute con- 
tro]. The unskilled or careless worker in the laboratory, who 
allows micro-organisms to escape from him while he is attempt- 
ing to study them, is a serious menace not only to himself but to 
all others in the laboratory. These two primary essentials must 
be mastered by practice in handling harmless forms. 

Every instrument with which bacteria are handled should be 
sterilized before it is used, and again after use. In the case of 
the commonly used platinum wire, this sterilization is accom- 
plished in the flame. The wire is heated to a glow and allowed 
to cool before handling bacteria, and immediately after its use, 
before it leaves the hand, it is brought close to the flame so as to 
dry the material on it and then again heated to redness. Care- 
ful drying in this way avoids sputtering and consequent scattering 
of bacteria, which is almost certain to occur if moist material, 


especially fat or protein, is carelessly thrust into the flame. 
108 


THE CULTIVATION OF MICRO-ORGANISMS Iog 


In using the Bunsen flame for sterilization, the innermost cone 
near the base of the flame may be utilized for drying material on 
the end of the wire. This inner cone is not burning and is com- 
paratively cool, and after a little practice the end of the wire 
is easily brought into it and dried without sputtering. Slowly 
elevating the wire brings it gradually into hotter zones of the 
flame until it glows. 

Bacteria do not of themselves leave a moist surface. They 
are not even removed by moderate currents of air unless they 
have been previously dried. Their distribution about the labora- 
tory, therefore, results from relatively gross accidents or gross 
carelessness. When material containing bacteria is accidentally 
spilled, it should be covered at once with disinfectant solution 
such as 1-1000 mercuric-chloride solution. As a routine pro- 
cedure it is well to wash the work table daily with bichloiide 
solution and, when working with pathogenic bacteria, to wash 
the hands at the end of the day’s work, first with the bichloride 
solution and then with scap and water. 

Isolation of Bacteria.—In order to study any kind of bacteria 
it is necessary to have the particular species separated from other 
sorts with which it may be mixed. The earlier bacteriologists 
endeavored to separate bacteria of different sorts by successive 
transplantations through a series of tubes of fluid media, one 
kind of bacteria outgrowing the rest. Isolation was also accom- 
plished by diluting the material very highly and then inoculating 
one drop into each of a large number of tubes of broth. Some 
tubes would thus receive no bacteria, others would receive several, 
and occasionally one would receive only a single germ and would 
give rise to a pure culture. Another early method of separating 
a pathogenic species was by inoculation of animals. The ability 
of the animal to prevent the development of all but one species 
contained in the inoculated material was utilized to obtain the 
first pure cultures of anthrax bacilli and tubercle bacilli. These 
methods are successfully employed only for relatively few bac- 


terial species. 


IIO BACTERIOLOGY 


Methods of isolating bacteria, which are of more general appli- 
cation, were introduced by Koch. The essential characteristic 
of these mehods is the dilution of the bacteria in a fluid medium 
which quickly becomes solid so that each germ develops in a 
definite fixed position in the medium. 

It is impossible in most cases to distinguish between bacteria 
of different varieties by microscopical examination alone. Bac- 
teria of widely different species and quite unlike one another in 
their properties may present similar appearances under the mi- 
croscope. The differences which they exhibit are usually appar- 
ent when they are grown in culture-media. The growth, called 
a colony, which results from the multiplication of a single 
bacterium, is in many cases very characteristic for the species. 
By the plate-method, the individual bacteria in a mixture are 
separated from one another by dilution. They are fixed in place 
by the use of a solid medium. They are allowed to grow, and 
from each individual there arises a colony. It is usually possible 
to distinguish between colonies arising from different species when 
it is not possible to distinguish between the individual bacteria 
of these species. A convenient comparison has been suggested by 
Abbott. A number of seeds of different sorts may appear very 
much alike, and considerable difficulty may be found in distin- 
guishing one from another with the eye. Let them be sown, how- 
ever, and let plants develop from them, and these plants will easily 
be distinguished from one another. 

Method of Making Plate-cultures.— Melt three tubes of gela- 
tin or agar. (There is some difficulty in keeping agar in a fluid 
state while dilutions are being made. It is convenient to have 
some form of water-bath with a thermometer for the purpose.) 
Let the liquefied agar cool to 45° C. Gelatin may be used ata 
temperature anywhere between 28° and 4o” C. Take a small 


1It must be understood that no close comparison can be drawn between higher 
plants, which simply complete the development of parts potentially present in the 
seed, and colonies of bacteria, which are aggregates of individuals, the progeny of one 
individual of the same kind. 


THE CULTIVATION OF MICRO-ORGANISMS Ill 


portion of the material to be examined—pus, for example—and 
introduce it with a sterilized platinum wire or loop into one of 
the tubes. The plug of the test-tube is to be withdrawn, twisting 
it slightly, taking it between the third and fourth fingers of the 
left hand, with the part that projects into the tube pointing to- 
ward the back of the hand. It must not be allowed to touch 
any object while the inoculation is going on. Pass the neck of 
the tube through the flame. If any of the cotton adheres to the 
neck of the tube, pull the cotton away with sterilized forceps, 
while the neck of the tube touches the flame, so that the threads 
of cotton may be burned and not fly into the air of the room. 


Fic. 41.—Method of inoculating culture media. 


The tube is held as nearly horizontal as possible, in the left hand 
between the thumb and forefinger, resting upon the palm, and the 
neck of the tube pointing upward and to the right. Mix the 
’ material introduced thoroughly with the liquefied culture-medium, 
taking care not to wet the plug. Now remove the plug again, 
and, having sterilized the platinum wire, insert it into the liquefied 
medium. Carry three loopfuls in succession from this tube, 
which is No. 1, into tube No. 2. When two tubes are being used 
at the same time, they are placed side by side between the thumb 
and forefinger of the left hand. The two plugs are held between 
the second and third and the third and fourth fingers of the left 
hand, respectively. The wire may now be passed into the first 
tube, which we will suppose to hold some material containing 
bacteria, and a little of this material may be removed on the tip 


II2 BACTERIOLOGY 


of the wire from the first tube to the second. When the needle 
is introduced into or removed from either tube it should not touch 
the side of the tube at,any point, and should only come in contact 
with the region desired. After inoculation of the second tube 
has been effected, the wire is heated to redness in the flame, the 
necks of the tubes are passed through the flame, and the plugs are 
returned to their respective tubes. In the same manner transfer 
three loopfuls from tube No. 2 into tube No. 3. The original 
material will obviously be diluted in tube No. 1, more in tube No. 2, 
and still more in tube No. 3. The most convenient form of plate 
is that known as a Petri dish, a small glass dish about 10 cm. in 
diameter and 1.5 cm. in height, provided with a cover which is 
a little larger but of the same form. This dish should be cleaned, 


Fic. 42.—Petri dish. 


dried and sterilized for an hour in a hot-air sterilizer at 150° C. 
or higher. When it is cool it may be used. 

Such dishes having previously been prepared, the contents of 
tube No. 1 are poured into one dish, and those of tube No. 2 
into another, and those of tube No. 3 into a third. They are 
labeled Nos. 1, 2, and 3.1. In pouring proceed as follows: re- 
move the plug of tube No. 1; heat the neck of the tube in the 
flame; allow it to cool, holding it in a nearly horizontal position. 
When the tube has cooled, lift the cover of the Petri dish a little, 
holding it over the dish; pour the contents of tube No. 1 into the 
dish, and replace the cover. The interior of the dish should be 
exposed as little and as short a time as possible. Tubes Nos. 2 and 
3 are to be treated in the same manner. Burn the plugs, and 

* The labels should be moistened with the finger, which has been dipped in water. 


They should not be licked with the tongue. While working in the bacteriological 
laboratory it is best to make it a rule that no object is to be put in the mouth. 


THE CULTIVATION OF MICRO-ORGANISMS 113 


immerse the empty tubes in 5 per cent solution of carbolic acid. 
Where much culture work is being done, it will be found convenient 
to sterilize the mouth of each tube by thorough heating in the flame 
after pouring out its contents, and then to replace the plug. 
The tube may then be placed in a special receptacle which is 
sterilized with its contents in the autoclave at 120° C. for 20 
minutes, at the end of the day’s work. 


Fic. 43.—Colonies in gelatin plate showing how they may be separated and the 
organisms isolated. 


The culture-medium in the Petri dish will soon solidify. 
Petri dishes of agar should be inverted after the medium is firmly 
set; otherwise the water, which evaporates from the surface and 
condenses on the inside of the lid, may overflow the surface of 
the agar, confusing the result. Agar plates are usually developed 
in the incubator. Gelatin plates must be developed at a tempera- 
ture below the melting-point of the medium, which is usually 
between 22° and 28° C. Colonies usually appear in from one to 
two days. In plate No. 1 they will be very numerous, in plate 
No. 2 less numerous, and in plate No. 3 still less numerous. 


Where the number is small the colonies will be widely separated 
8 


114 BACTERIOLOGY 


and can readily be studied. They may be examined with a hand- 
lens, or the entire dish may be placed on the stage of the micro- 
scope and the colonies be inspected with the low power. The 
iris diaphragm should be nearly closed and the plane mirror should 
be used. Dilution-cultures prepared as described in the next 
paragraph, where the principle is the same, are shown in Fig. 45. 
In tube No. 1 the colonies are so numerous as to look like fine white 
dust. In tubes 2 and 3 they are less numerous and larger. 
Esmarch’s Roll-tubes.—Use liquefied ‘gelatin or agar. The 
dilutions in tubes 1, 2 and 3 are made asabove. Tubes contain- 
ing a rather small amount of the culture-medium are more con- 


Fic. 44.—Manner of making Esmarch roll-tube. 


venient. A block of ice should be at hand, and, with a tube filled 
with hot water and lying horizontally, a hollow of the size of the 
test-tube should be melted on the upper surface of the ice. In 
this hollow, place the tube of liquefied gelatin or agar; roll it rapid- 
ly with the hand, taking care that the culture-medium does not 
run toward the neck as far as the cotton plug. The medium is 
spread in a uniform manner around the inside of the tube, where 
it becomes solidified. Gelatin rcll-tubes must be kept in a place 
so cool that there is no danger of their melting; in handling them 
they are to be held near the neck, so that the warmth of the hand 


THE CULTIVATION OF MICRO-ORGANISMS 11S 


may not melt the gelatin. Agar roll-tubes should be kept in a 
position a little inclined from the horizontal, with the neck up, 


Fic. 45.—Dilution-cultures in Esmarch roll-tubes. In tube 1 the colonies are 


very close together; in tube 2 they are somewhat separate; in tube 3 they are well 
isolated. — | 


for twenty-four hours, so that the agar may adhere to the wall 
of the tube. 


x 


116 BACTERIOLOGY 


In the plate-method as originally devised by Koch, instead of using 
Petri dishes, the gelatin was poured upon a sterile plate of glass. This plate 
of glass was laid on another larger plate of glass, which formed a cover for a 
dish of ice-water, the whole being provided with a leveling apparatus. ‘The plate 
was kept perfectly level until it had solidified,’ which took place rapidly on the 
cold surface. The glass plates were placed on little benches enclosed within a 
sterile chamber. The more convenient Petri dish has now displaced the original 


glass plate. 


Streak Method of Isolating Bacteria.—The isolation of bac- 
teria may sometimes be effected by drawing a platinum wire 
containing material to be examined rapidly over the surface of a 
Petri dish containing solid gelatin or agar; or over the surface of 
the slanted culture-medium in a test-tube; or by drawing it over 
the surface of the medium in one test-tube, then, without steril- 
izing, over the surface of another, perhaps over several in succes- 
sion. This method is ordinarily less reliable than the regular 
plating method. 

Veillon’s Tall-tube Method.—Three to six tubes of glucose 
agar, the agar being at least 6 cm. deep, are liquefied and cooled 
to 45° C. in a water-bath. A small amount of the material to 
be examined is placed in the first tube by means of the platinum 
loop, and carefully mixed. From this dilutions are made in series 
to tubes 2, 3, 4,5 and 6, each being carefully mixed without intro- 
ducing airbubbles. The tubesare quicklysolidified by immersion 
in cold water, and are incubated at 37° C. These culture tubes 
offer the contained bacteria a wide range of oxygen supply. This 
is abundant at and near the top, and gradually diminishes lower in 
the tube until near the bottom almost perfect anaérobic conditions 
obtain. The method is very useful in isolating B. bifidus from 
feces of infants, B. acne from acne pustules, and in studying the 
oxygen requirements of other bacteria and ‘has been most exten- 
sively employed for study of the bacteria in war wounds, where it 
has proved to be of fundamental importance. Colonies are picked 
out with sterile glass capillaries, and deeper colonies are reached 
by breaking the tube. The successful use of the method requires 
some practice and particular care, both in the preparation of 


THE CULTIVATION OF MCRO-ORGANISMS airy 


the agar and the cultivation of bacteria, is essential to success in 
studying toxic edema and gaseous gangrene of war wounds. 

Appearance of the Colonies.—The colonies obtained in the 
Petri dishes or in tubes (Fig. 45) may be studied with a hand- 
lens or with a low power microscope. In the latter case, use the 
plane mirror with the iris diaphragm nearly closed. The colonies 
present various appearances. Some of them are white, some 
colored; some are quite transparent and others are opaque; some 
are round, some are irregular in outline; some have a smooth 
surface, others appear granular, and others present a radial 
striation. Surface colonies often present different appearances 
from those occurring more deeply. Surface colonies are likely 
to be broad, flat and spreading. If the colony consists of bacteria 
which have the property of liquefying gelatin, a little funnel- 
shaped pit or depression forms at the site of the colony. The 
appearance of colonies may be of great assistance in determining 
the character of doubtful species. The appearance in gelatin 
plates of the colonies of the spirillum of Asiatic cholera, for in- 
stance, is one of the most characteristic features of this organism. 

Pure Cultures.—From these colonies pure cultures may be 
obtained by the process called ‘‘fishing.”” Select a colony from 
which cultures are to be made, touch it lightly with the tip of a 
sterilized platinum wire, taking great care not to touch the medium 
at any other point. Introduce the wire into a tube of gelatin 
after removing the plug and flaming the mouth of the tube. 
Sterilize the wire and plug the tube. In a similar manner, and 
from the same colony, inoculate tubes of agar, bouillon, milk, 
potato and blood-serum. Gelatin tube cultures are usually 
inoculated by introducing the platinum needle into the medium 
vertically, making a “‘stab-culture.”’ Inclined surfaces such as 
those of agar, potato or blood-serum are inoculated by drawing 
the wire lightly over the surface of the medium, making a “smear- 
culture” or ‘“‘streak-culture”’ (Figs. 46 and 47). Liquid media are 
inoculated by simple introduction of a small mass of bacteria and 
mixing them with the medium. At the same time it is well to 


118 BACTERIOLOGY 


make a smear preparation from the colony and to stain with one 
of the aniline dyes so as to determine the morphology of the 
bacteria. The growths which take place in the tubes should con- 
tain one and the same kind of bacteria. As seen under the micro- 
scope these- bacteria should have the same general form and 
appearance as those seen in the colony from which they were 


Fic. 46.—Stab-culture. Fic. 47.—Smear-culture. 

A rubber stopper may This tube shows the 

*be used to prevent drying, rubber cap used to prevent 
see page I19. drying. 


derived. This will be the case, provided the colony has resulted 
from the development of a single bacterium. 

A pure culture is a culture which contains only the descendants 
of a single cell. 

Stock Cultures.—To maintain their vitality bacteria need to 
be transplanted from one tube to another occasionally; the time 
varies greatly with different species. Many bacteria grow on 
culture-media with difficulty at the first inoculation, but having 


THE CULTIVATION OF MICRO-ORGANISMS IIQ 


become accustomed to their artificial surroundings, as it were, 
they may be propagated easily afterward; this is especially true 
of the tubercle bacillus. After they are developed, stock cultures 
are best kept in a refrigerator, and it is well to seal them so as to 
prevent drying. Rubber caps or rubber stoppers are useful for 
this purpose (Figs. 46 and 47). 

Some kacteria flourish better on one culture-medium than 
another. The tubercle bacillus grows best on blood-serum and 
glycerin-agar; the bacillus of diphtheria grows best on Léffler’s 
blood-serum; the gonococcus on human sérum-agar or ascitic- 
fluid-agar. 

The virulence of most pathogenic bacteria becomes diminished 
after prolonged cultivation upon media. In some forms the viru- 
lence is lost very quickly, for example, the streptococcus and 
pneumococcus. 


REGULATION OF TEMPERATURE 


High-temperature Incubator—Many bacteria flourish best 
at a temperature about that of the human body, 37° C. Some 
species will grow only at this temperature. The pathogenic bac- 
teria in particular, for the most part, thrive best at a point near 
the body temperature. 

The ordinary incukator is a box made of copper, having 
double walls, the space between the two being filled with water. 
‘The outer surface is covered with some non-conductor of heat, 
such as felt or asbestos. At one side is a door, which is also double. 
The inner door is of glass, the outer door is of copper covered 
with asbestos. At one side is a gauge which indicates the level 
at which the water stands in the water-jacket. The roof is per- 
forated with several holes, some of which permit the circulation 
of the air in the air-chamber inside the box; some of them enter 
the water-jacket. A thermometer passes through one of these 
holes into the interior of the air-chamber, and often another into 
the water standing in the water-jacket. A gas-regulator passes 
through another hole, and is immersed in the water standing in 


120 BACTERIOLOGY 


the water-jacket. There are various forms of gas-regulators 
more or less complicated. The simplest and least expensive 
thermo-regulators for gas are made of glass and filled with mercury 
or with mercury and some lighter liquid, the heavy mercury 


WY 
\ 


Fic. 48.—Incubator. 


serving to close the chief source of gas supply when the desired 
temperature has been attained, while a minute opening at another 
point remains open to furnish sufficient gas to keep the flame 
alight, but not sufficient to maintain the temperature. Upon 


, 


‘ imperfectly shut off at the desired temperature, 


THE CULTIVATION OF MICRO-ORGANISMS 121 


cooling the mercury falls and allows gas to flow again through 
the larger opening. In this way the supply of gas is made large 
whenever the temperature is a little below the desired temperature 
and very small whenever the temperature rises above that point, 
and the temperature varies within a slight range. The Reichert 
regulator is designed to operate according to 
these principles, and various modifications of 
this regulator are on the market. In many 
of these instruments the larger supply is only 


and, where the weight of the mercury is relied 
upon to stop this opening, the gas may often 
bubble out through it unless special precau- 
tions are taken to regulate the pressure of the 
gas supply... 

A modification of this type of regulator 
devised by Mac Neal! overcomes this difficulty 
(see Fig. 50). The inlet tube A leads through 
the wall of the chamber D, to which it is fused, 
into an inner upright tube, BC. Near the 
upper end of this upright is a small opening, 
O, which allows the minimum supply of gas Fic. 49.—Reichert’s 
to pass to the burner to avoid extinction of the 8s Teewlator- 
flame. The lower end of this upright tube fits quite closely the bot- 
tom of the chamber D, around the opening leading into the capillary 
tube, EF. This end is adjusted so close to the bottom that mer- 
cury will nct pass through. between inner and outer tube at less 
that twenty millimeters mercury pressure, yet not so close but 
than an abundant supply of gas may"pass. The proper adjust- 
ment of this part must be throughly tested before the instrument 
leaves the factory. The upper end of the upright, BC, is closed 
by a ground glass stopper, which also closes the top of the outer 
chamber, D. In the ground surface of this stopper a gamma- 
shaped (I) groove is cut, the vertical limb extending from the 

1 The Anatomical Record, August, 1908, Vol. I1, No. 5. 


122 BACTERIOLOGY 


lower tip of the stopper to the level of the opening, O. The 
horizontal limb is deep where it joins the vertical, but gradually 
becomes shallow and ends about one-quarter the way around the 
stopper. This groove serves for passage of the gas from the inner 
tube BC, to the opening O, and thus to the 
outer chamber D, and by rotating the stopper, 
the amount of gas flowing through this 
passage may be reduced to any desired point. 
The outlet tube, H, leads from the chamber D 
to the burner connection. 

The capillary, EF, leads to a bulb of suffi- 
cient size; the larger the more sensitive the 
instrument. Either the large bulb with in- 
side capillary, J, to be filled with mercury 
and toluol, or the smaller simple bulb for 
mercury alone may be used. A side arm is 
attached to one side of the capillary EF, for 
conveniently controlling the height of the 
mercury column. Fither the curved capillary 
\ tube with stopcock and a cup on the end, or 
the simple tube with metal screw cemented 
in, may be used here, according to the pur- 
} pose which the regularor is to serve. These 
parts are similar to those of Novy’s modi- 
fication of the Reichert regulator. 

To fill the instrument, the air is partly 
U driven out by heating the bulb ‘and then the 
B aa desired liquid is drawn in by cooling, re- 

peating the heating and cooling until the 
instrument is full of the liquid. For the small bulb, mercury is 
always used alone. The large bulb, on the other hand, is filled 
first with’ either ether, alcohol or toluol, and then part of this 
liquid is forced out by heat and replaced with mercury so that 
the capillary EF, the bulb at its lower end, and a small part of the 
large bulb'J, are occupied by the mercury. Ether may be used 


bad 


e 
THE CULTIVATION OF MICRO-ORGANISMS 123 


when the regulator is not to be heated above 35° C., alcohol when 
itis not to be heated above 75° C., and toluol for temperatures 
between 75° and 100° C. 

A very satisfactory regulator is that of Roux. It is con- 
structed entirely of metal, and its operation is due to the unequal 
expansion and contraction of two 
metals which are riveted together. 
Fig. 51 shows this regulator. The 
gas passes in at e and passes out 
atd. The amount of gas passing 
through is regulated by a piston 
on the end of the set screw inside 
the tube from which the outlet 
tube branches cff. This piston 


Fic. 52.—Koch automatic gas- 
regulator. a, Set screw; b, Screw burner, 


collar; c, Clamp; d, Outlet for 

gas; e, Inlet for gas. 
moves in or out according to the changes of temperature of the 
water jacket of, the incubator into which the stem (f) of the regu- 
lator is inserted. This stem is fenestrated and has the riveted 
metallic strips running down in it. These strips are pivoted at 
the collar, g. 

The gas coming from the gas-regulator passes to a Bunsen 
burner, which stands underneath the incubator. This burner 
should have some kind of automatic device for cutting off the flow 
of gas in case it becomes accidentally extinguished by a sudden 


124 BACTERIOLOGY 


draught of air or from any other cause. The automatic burner 
invented by Koch is an ingenious, simple and effective device 
(Fig. 52). The coils of metal seen on each side at the top of the 
burner are so arranged that when they expand they turn the 
disk below so as to support the arm coming from the stop-cock; 
when they cool they turn the disk in the opposite direction, and 
allow the arm to fall and cut off the gas. Some inconvenience 
will at times arise from irregularities in the flow of gas from the 
main supply-pipe. A properly constructed regulator should, 
however, compensate perfectly for all ordinary variations in pres- 
sure of artificial gas. Natural gas is commonly furnished at 
much higher pressure and it is necessary to install apparatus 
to reduce the pressure, a gas-pressure regulator, between the gas 
main and the thermoregulator. Fluctuations of the temperature 
within the incubator depend very largely. upon the external 
temperature, especially if its outer walls are not well insulated. 
The incubator should, therefore, be kept in a place free from 
draughts of air, where the temperature is fairly constant. 

In large modern laboratories, the incubators are built in as 
special insulated rooms, heated by a gas stove. A regulator of 
large size is installed to control the supply of gas to the stove. 
These incubator rooms are very satisfactory and provide quite a 
range of constant i aa according to the height of shelves 
from the floor. 

Culture-tubes which are sath kept in the incubator are likely 
to become dry if their stay is prolonged. In such cases they 
should be covered with rubber caps, tin-foil, sealing-wax, paraffin, 
or some other device to prevent evaporation. If rubber caps 
are used, they should be left in 1-1000 bichloride of mercury 
solution for an hour, and the cotton plugs should be singed in the 
flame, before putting them on (Fig. 47). Some bacteriologists 
prefer rubber stoppers, which may be boiled and stored in bi- 
chloride of mercury solution. Cut the cotton plug even with the 
edge of the tube; singe it in the flame; push it into the tube about 
1 cm., and insert the rubber stopper (Fig. 46). 


THE CULTIVATION OF MICRO-ORGANISMS 125 


Low-temperature Incubator.—An incubator regulated for so- 
called “room temperature” is very desirable for the cultivation 
of bacteria upon gelatin and for the bacteriological analysis 
of water. In our climate the temperature of the rooms of the 
laboratory often reaches a point at which gelatin melts, and 
for this reason in a low-temperature incubator provision has to 
be made for cooling when the room temperature is too high as 
well as for heating when it is too low. 

A form of incubator devised by Rogers! for this purpose 
consists of a refrigerator or of a specially constructed chamber 
heated by electricity and controlled by an electric thermoregu- 
lator. Below is given a description of an incubator constructed 
according to Rogers’ plans. This incubator has been in use: 
for some time and has given entire satisfaction since the precautions 
noted below were followed. There would appear no reason 
why this incubator should not be employed for high temperatures 
as well as for low, but so far it has been run at 22° C. The tem- 
perature has kept very constant. The incubator consists of a 
refrigerator, 30 inches high, 24 inches wide, 18 inches from front 
to back, all outside measurements. Instead of the ordinary 
drip pipe, there is a coil of 1-inch galvanized iron pipe run down 
the back of the cooling chamber attached water-tight to the ice 
tank. From the bottom of the cooling chamber the coil runs up 
perpendicularly nearly to the bottom of the ice compartment, 
and then runs horizontally through the wall of the refrigerator. 
A bracket on the outsidé supports a drip-pan. A thermometer 
encased in a fenestrated metal jacket is inserted ‘about half 
way up on oneside. A lump of ice, about 50 pounds, placed in the 
ice compartment serves to keep the temperature sufficiently 
cool. In summer doubtless more ice will be required. 

For heating, an ordinary 16-candle-power electric bulb is 
used, and the electricity is obtained from the public supply. 
The wire is run through one of the walls, and a part of the current 


1L. A. Rogers. On electrically controlled low temperature incubators. Cen- 
tralblatt fiir Bakteriologie, etc., Bd. XV, Abt. LI, pp. 236-239, Sept. 23, 1905. 


126 BACTERIOLOGY 


is made to operate a horse-shoe magnet, and another part is 
conducted through the lamp used for heating. 

The accompanying diagram (Fig. 53), will serve to show the 
arrangement. 

A telegraph key is used to supply the horse-shoe magnet 
which is inserted in the heating circuit in such a way that when 
the armature is attracted toward the magnet the circuit is com- 
pleted and the lamp is consequently lighted. The part of the 
current, a, supplying the magnet first passes through a small 


Resistance -cot/s mY 
om 4 
ee 


Reastance 7 
Lamp 
ec to 
a = 
ee a . | 
= Heating Lomp 


=f 


a <— Brasa 
mei 1.7: ee ae 
Thermoregulator 


Fic. 53.—Diagram of electric regulator for low-temperature incubator. 


lamp and through two resistance coils so as to reduce the current. 
It then passes through the magnet, and is continued on to the 
set-screw, 6, which is so placed that when the thermoregulator 
comes in contact with it the circuit is.complete. The regu- 
lator consists of a strip of hard rubber and a strip of brass riveted 
together. One end is fixed, while the other is free, and when it 
is heated it tends to bend toward the metal side, when it cools it 
bends toward the rubber. The brass strip is 15 inches long, 4 
inch thick, and 14 inch wide; the rubber strip is 14 inch thick, 4 
inch wide, and a little less than 15 inches long. In the diagram 
the end is fixed at d and is free at b. When it is heated, the free 


THE CULTIVATION OF MICRO-ORGANISMS 127 


end travels away from the set-screw at b; when it cools, it moves 
toward the set-screw. Rogers also recommends a regulator 
made of invar and brass instead of hard rubber and brass. Where 
invar is used instead of the hard rubber the dimensions for the 
two metals are the same as those given for the brass strip in the 
hard-rubber-brass regulator just described. As is evident from 
the description, the circuit controJling the magnet is closed when- 
ever the free end of the regulator comes in contact with the set 
screw at 6. When this circuit is closed the magnet attracts the 
armature, and the heating circuit is closed by the contact formed 
at c between the armature and the set-screw. In thediagram 
this point of contact is put to one side for the sake of clearness, 
but as a matter of fact in the instrument in use, the set-screw is 
above and between the ends of the horse-shoe magnet, and comes 
in contact with the armature which is extended upward in the 
shape of a tongue. From the description just given it will be 
noted that the thermoregulator does not control the heating 
directly, but indirectly through the electro-magnet. 

Certain precautions have been found necessary in practice 
in order to obtain satisfactory results with this incubator. The 
set-screw against which the armature strikes at c should be so 
set that the armature does not come in contact with the magnet. 
In the apparatus described above there is a space of about 14 inch 
between the armature and the magnet when contact takes place 
between the set-screw and the armature. If the set-screw does 
not project far enough to prevent the armature from coming in 
contact with the magnet, the armature may adhere to the magnet 
even after the current is broken at 6, and when this is the case of 
course the lamp remains lighted, and the temperature may run 
up too high. This sticking of the armature to the magnet is 
said to be due to the residual magnetism left in the core of the 
magnet. When the current passing through the magnet is 
broken by the excursion of the end of the thermoregulator away 
from the set-screw at 6, the armature is pulled away from the 
magnet by a coiled spring. Another important precaution is 


128 BACTERIOLOGY 


that the points at which contact is made and broken, 0 and c, 
should be tipped with platinum. A small piece of platinum 
wire inserted into the ends of the set-screws and a few square 
centimeters of platinum foil riveted to the opposite point of con- 
tact, meet the requirements. If platinum is not used at these 
points oxidation takes place and prevents contact. The set- 
screw at b is set by experiment for the temperature desired. 
The further the point of the set-screw. projects toward the free 
arm of the regulator, the higher the temperature maintained. 

Electrically heated and electrically regulated incubators for 
any desired temperature are now to be found on the market. 
Their initial cost is rather high and, as a rule, they are adapted - 
for use with only one kind of electric current. The exact current 
available should be stated in ordering. 

In many places electric current is not constantly available and 
in the field one often has to work without gas. Highly satisfactory 
oil heated, water-jacketed incubators came into very general use 
in field laboratories in England and’France during. the war. 


CULTIVATION OF ANAEROBIC BACTERIA 


Deep Stab Culture.—Bacteria which cannot grow in the pres- 
ence of atmospheric oxygen may be successfully cultivated by 
methods in which the oxygen is excluded or its concentration 
diminished. The simplest procedure, first practised by Liborius, 
is to make deep stab cultures into freshly solidified alkaline 
glucose agar. The agar quickly closes over the needle track and 
any traces of oxygen introduced into the depths of the agar are 
absorbed and reduced by the glucose in the presence of the 
alkali. The bacteria thus find at various points along the punc- 
ture all variations in partial pressure of oxygen from almost 
complete absence up to the concentration existing in the atmos- 
phere at the surface of the medium. Obligate anaérobes begin 
to grow near the bottom and, as the gases produced replace the 
air above, the growth extends upward, often even entirely to the 
surface. 


THE CULTIVATION OF MICRO-ORGANISMS 129 


Veillon Tube Cultures.—Isolated colonies of anaérobic bac- 
teria may be obtained by a modification of this tube method of 
Liborius, which seems to have been used first by Veillon. The 
principle of the method has been given on page 116. 

Fermentation Tube.—Anaérobic bacteria grow excellently 
in the Smith fermentation tube filled with glucose broth, especially 
if a small piece of naturally sterile liver or kidney from a small 
animal, or a few cubic centimeters of naturally sterile defibrinated 
blood be added to the medium in the tube. Glucose gelatin 
to which litmus has been added also furnishes a medium in which 
anaérobes will grow abundantly without any special precautions 
to protect them from oxygen or from the air. 

Removal of Oxygen.—Anaérobic conditions may be furnished 
by pumping out the air from a container in which the cultures 
have been placed, a method employed by Pasteur. The oxygen 
may be absorbed from the air by a mixture of pyrogallic acid 
and alkali. Buchner’s method is carried out as follows: Into 
a bottle or jar, which can be tightly stoppered, pour ro c.c. of a 
6 per cent solution of sodium or potassium hydroxide, for each 
100 ¢.c. of air contained in the jar. Add one gram of pyrogallic 
acid for each 10 c.c. of solution. The culture-tube is placed 
inside of the larger bottle or jar, supported above the bottom, 
and the stopper, smeared with paraffin, is inserted. The mix- 
ture of pyrogallic acid and potassium hydroxide possesses the 
property of absorbing oxygen. 

Wright’s Modification of Buchner’s method: The tube of cul- 
ture-medium is plugged with absorbent cotton, using a plug of 
large size. The culture-medium is inoculated in the usual way. 
The plug is cut off close to the neck of the tube, and is then pushed 
into the tube about 1 centimeter. Now allow a watery solution 
of pyrogallic acid to run into the plug, and then a watery solution 
of sodium or potassium hydroxide. Close quickly and tightly 
with a rubber stopper. Wright recommends that the first 
solution be freshly made and consist of about equal volumes of 


pyrogallic acid and water, and that the second solution contain 1 
9 


130 BACTERIOLOGY 


part of sodium hydroxide and 2 parts of water. With 6 inch test- 
tubes, 34 inch diameter, the amounts advised are }4 c.c. solution 
of pyrogallic acid and 1 c.c. solution of sodium hydroxide. 
Hydrogen Atmosphere-—The most 
==: perfect anaérobic conditions are ob- 
tained by replacing the air with hydro- 
i gen in a perfectly air-tight container. 
The method of hermetically sealing 
Hee such containers full of hydrogen by 
a melting the glass in a flame is really 
a too dangerous to be recommended. 
The apparatus devised by Novy is 
most convenient and has practically 
superseded all other devices for culti- 
vation of anaércbes in hydrogen. The 
Novy jar is especially valuable for plate 
cultures. In using this jar, all ground- 
glass surfaces should be thoroughly 
coated with a fairly stiff mixture of 
bees wax and olive oil so as to make all 
joints air-tight. Rubber gascots or 
packing should never be employed ke- 
tween the ground-glass surfaces, re- 
Fic. 54.—Arrangement of gardless of the fact that many dealers 
eS ee furnish them for this purpose. After 
the plate cultures or tubes have been 
put into the lower section of the jar, the cover is put on so that 
the flanges fit together perfectly. A heavy rubber band may 
then be passed around the circumference of the flanges to cover 
the circle of contact. F inally two or three clamps, the jaws of 
which are cushioned with cork or with rubber, are fastened on 
the flanges, pressing them firmly together. The jar is now 
attached to a source of pure hydrogen so that the gas enters at 
the top of the jar. The other opening is connected with a wash 
bottle containing water which serves as a valve. Hydrogen is 


| == 
— 
t 
\ 
‘= 


Ty 
anean 


THE CULTIVATION OF MICRO-ORGANISMS I31 


Fic. 55.—Bottle for tube cultures. 


Mu, 


(After Novy.) 


Fic. 56.—Apparatus for Petri dishes or tubes— 
gas or pyrogallate method. (After Novy.) or 


Fic. 57.—Apparatus for plates 
tubes—gas, pyrogallate or vac- 


uum method. (After Novy.) 


132 BACTERIOLOGY 


passed through the jar for two hours or more. It is well to keep 
all flames away from the apparatus as a precaution against ex- 
plosion of the hydrogen when mixed with air. 


EN Be 


Fic. 58.—Tripod and siphon flask for anaérobic culture by combined hydrogen and 
pyrogallate method. 

The hydrogen is generated by the action of 25 per cent sul- 

phuric acid on granulated zinc. It should be purified by passing 

through a wash bottle of alkaline lead acetate solution, a second 


THE CULTIVATION OF MICRO-ORGANISMS, 133 


one containing a solution of potassium permanganate and a 
third of silver nitrate. In diluting sulphuric acid, the acid 
must be poured slowly into the water, and the mixture cooled in 
a bath of cold water, or under the tap. Carelessness in dilut- 
ing this acid may allow violent boiling to occur, sometimes with 
serious consequences. 


Fic. 59.—An aérobic organism (potato coe that will not grow under a cover- 
glass. 

For critical work in anaérobic culture it is well to combine 
the pyrogallate and hydrogen methods. This is readily accom- 
plished by placing the Petri dishes on a low glass tripod with a 
small amount (2 grams) of pyrogallic acid beneath them on the 
bottom of the Novy jar.!. On top of the stack of Petri dishes is 
placed a small flask containing strong solution of sodium hydrox- 
ide, and provided with a siphon spout (see Fig. 58). A rubber 
tube is attached to this spout and leads down to the floor of the 
jar. After hydrogen has been passed through the jar and it has 
been finally closed, a slight tipping to one side starts the flow of 
the alkali through the siphon and so makes the pyrogallic acid 
available to absorb the last traces of oxygen. 

a MacNeal, Latzer and Kerr, Journ. Infect. Diseases, 1909, Vol. VI, p. 557. 


134 BACTERIOLOGY 


Further Anaerobic Methods.—Numerous other expedients 
have been employed for the cultivation of anaérobes. Koch 
covered part of the surface of a gelatin plate with a bit of steril- 
ized mica or a cover-glass. Such a method suffices to prevent the 
growth of strictly aérobic forms but rarely suffices for the success- 
ful culture of strict anaérobes. Covering the surface of the 
medium with sterile liquid paraffin is a more perfect means of 
excluding air. 

In all anaérobic culture methods, the presence of one or more 
reducing substances in the culture medium is of great importance. 
Those commonly employed are glucose, litmus and native protein. 


CHAPTER VI 
METHODS OF ANIMAL EXPERIMENTATION 


Value of Animal Experimentation—The importance of ex- 
perimentation upon animals in the development of our knowledge 
concerning disease-producing micro-organisms can hardly be 
over-estimated, and animals must be used in considerable numbers 
in any adequate presentation of the subject to a laboratory class 
in pathogenic bacteriology. Only in this way has it been possible 
to discover the causal relation of bacteria to disease and the way 
in which diseases are transmitted, and it is only by the use of 
animals that this information can be presented first-hand to 
students. The inoculation of animals also provides accurately 
controlled material for studying the course and termination of 
the disease as well as the gross or microscopic lesions produced 
by it. 

Care of Animals.—Laboratory animals should be housed in a 
light, well-ventilated room which should te heated in winter to 
about 60° F. If possible a run-way in the open air should be 
provided. The fixed cages may be constructed with wood or 
steel frames, but at least the front and preferably both front and 
back should be made of strong wire netting to provide ample 
ventilation. For rats and mice it is well to provide an enclosed 
perfectly dark space inside the cage into which these animals 
may retire. Smaller movable cages must also be provided for 
animals acutely sick and those infected with dangerously com- 
municable diseases. These must be sterilizable, and wood should 
not be used in their construction. Glass jars with weighted 
covers of wire netting are useful for mice and rats, and for larger 
animals such as guinea-pigs, rabbits and cats, cages of galvanized 
iron and wire netting are used. Pigecns may also be kept in such 

135 - 


136 BACTERIOLOGY 


cages. Very large animals, such as monkeys and dogs, require 
specially constructed cages. Laboratory animals should receive 
very careful attention. They should be supplied with new food 
at least once daily and with clean water twice a day. If food 
remains at the end of the day, it should be removed and a smaller 
amount given for the next day. The cages should be completely 
emptied and cleaned at least once a week, the refuse being in- 
cinerated. The animal house should be screened, and insects of 
all kinds given careful attention. It will be found practically 
impossible to control the lice and fleas, but winged insects, es- 
pecially biting varieties, may be kept out; and bedbugs, which 
sometimes gain entrance on new lots of guinea-pigs or rats, should 
not be allowed to remain uncontrolled. These possible carriers 
of infection require serious consideration as sources of confusion 
where experimental investigations are being carried out, not to 
mention the element of danger to the human individuals in the 
neighborhood. 

Holding for Operation.—Animals to be inoculated or operated 
upon must be held in a fixed position. Many special mechanical 
holders have been devised for the various animals, but these 
are nct necessary or especially useful. A pair of long-handled 
hemostatic forceps with lock, or a pair of placental forceps with 
lock, will be found most serviceable in handling mice or rats, 
the loose skin of the animal’s neck being caught in the forceps. 
Guinea pigs are best held by an assistant, the thumb and fore- 
finger of one hand encircling the thorax just behind the fore legs 
and the other hand helding the hind legs stretched out. Rabbits 
are held by the ears and hind legs with the body stretched over 
the knee. Monkeys are to be handled with thick gloves and 
should be caught around the neck from behind with one hand and 
by the pelvis or hind legs with the other. A second assistant 
is required to hold the fore legs. For all work which would cause 
any considerable pain the animal must be anesthetized, either 
by putting it into a closed compartment with the anesthetic or 
by use of a cone. Anesthesia is also necessary when delicate 


METHODS OF ANIMAL EXPERIMENTATION 137 


manipulations are to be carried out. For operations requiring 
some time the animal is fastened to a board with stout cords, or 
is held by means of a specially constructed animal holder. 

Inoculation.—Infectious material may be introduced into 
the animal body in various ways. The most common methods 
are injection under the skin and injection into the peritoneal 
cavity. The hair should be removed from the site selected. 
A sterilized hypodermic syringe is used, and it is again sterilized 
by boiling after use. Subcutaneous injection is usually made 
in the thoracic region as one easily avoids penetrating the chest 
cavity. For intraperitoneal injection the needle is quickly thrust 
through the abdominal wall. 

Inoculation into the cranial cavity is practised especially in 
studying rabies. The animal, rabbit or guinea-pig, is anesthe- 
tized and the scalp is shaved. An incision through the scalp 
about 8 to 10 mm. long is made at the left of the median line 
and parallel with it, a little in front of a line connecting the 
external auditory openings. The scalp is then forcibly drawn 
over to the right and a hole drilled through the skull at the right of 
the median line. A sharp-pointed scalpel may serve the purpose 
of a drill. The needle is then inserted into the cerebral substance 
nearly to the floor of the cranial cavity and the material (0.1 
to 0.5 c.c.) injected. Any blood or fluid is taken up with sterile 
absorkent cotton. The skin is replaced in its original position 
and may be dressed with cotton and collodion, although dressing 
may be omitted altogether. 

Inoculation inte the circulating blood is a method of special 
importance. -In rabbits intravenous injection is easily done. 
The hair is removed from the ear over the marginal vein, and 
the vein is dilated by application of a hot towel, after which the 
skin is wiped dry. An assistant constricts the base of the ear 
to congest the vein and the needle is easily inserted into it. Other 
veins on the ear may be used, but they are not so easily penetrated 
by the needle. In rats, guinea-pigs or monkeys, intravenous 
injection is not so simple and it is easier to inoculate these animals 


138 BACTERIOLOGY 


by intracardiac injection. For this purpose the animal is etherized 
and the precordial region is shaved and disinfected. The material 
to be injected is taken up into a Luer glass syringe. A second 
syringe, empty, with needle attached, is used to puncture the 
chest wall and the heart, preferably the wall of the right ventricle. 
The needle is introduced in the inter-costal space directly over 
the heart and near the border of the sternum. The appearance 
of blood in the previously empty syringe gives notice that the 
cavity of the heart has been entered. The syringe is now detached 
from the needle and the other syringe which contains the material 
to be injected is quickly substituted for it. The injection is made 
slowly. 

Other Sites for Inoculation—Many other regions are easily 
reached with the injection needle, such as the pleural cavity, the 
chambers of the eye, the spinal canal, the interior of muscles, 
and the substance of the testis. 

Subcutaneous Application.—Inoculation may be accomplished 
without using a syringe if desired. The skin and mucous mem- 
branes may be scratched with a needle or other instrument and 
the infectious material applied to the slight wound thus made. 
A small pocket may be made under the skin by making a small 
incision and introducing a blade of the forceps to separate the 
skin from the underlying muscle; and into sucha pocket one may 
introduce solid material, bacteria from a culture, pieces of tissue, 
garden soil or splinters of wood, with accompanying bacteria. 
The opening of the pocket is closed by cauterization or sealed 
with collodion. 

Alimentary and Respiratory Infection—Animals are some- 
times infected by feeding the virus, occasionally by injection 
into the rectum. Infection of the respiratory tract by spraying 
infectious material in the air breathed by the animal is rarely 
employed. 

Collodion Capsules.—Bacteria may be cultivated in the 
living body of an animal, without infecting the animal, when they 
are enclosed in collodion capsules. Their soluble products are 


METHODS OF ANIMAL EPXERIMENTATION 139 


able to diffuse through the collodion, while the animal’s fluids may 
pass into the sac to nourish them. Thése capsules were originally 
made by dipping the round end of a glass rod into collodion 
repeatedly. McCra’s method! is easier and more satisfactory. 
(Fig. 60.) 

A piece of glass tubing is taken, and a narrow neck drawn on it near one end. 
This end of the tube is rounded in the flame and, while still warm, the body of a 
gelatin capsule is fitted over it, so that the gelatin may adhere to the glass. The 
capsule is now dipped into 3 per cent collodion, covering the gelatin and part ot 
the glass. It is allowed to dry a few minutes, and is dipped again, In all, two or 


three coatings may be given. The capsule is filled with water and boiled in a 
test-tube with water. The melted gelatin is removed from the inside of the capsule 


U ae. 


Fic. 60.—Method of making collodion capsules. (After McCre.) 


i 


by means of a fine pipette. The capsule is partly filled with water or broth and 
sterilized. The capsule may now be inoculated. The narrow part of the glass 
tube which constitutes the neck must then be sealed in the flame, taking care that 
the neck be dry. The sealed capsule should be placed in bouillon for twenty-four 
hours. No growth should occur outside the capsule if it is tight. It may now be 
placed in the peritoneal cavity of an animal. 

A method of making collodion sacs recommended by Gorsline® consists in the 
use of a glass tube, the lower end of which is rounded and closed, except a small 
hole, which is temporarily filled with collodion. This tube is dipped in collodion 
and dried, as above. It may now he filled with water. By blowing at the opposite 
end, the pressure through the hole in the bottom of the glass tube will cause the 
capsule to loosen so that it comes away easily. Sacs made in this way are soaked in 
water for 30 minutes, dried and attached to the glass tube by gentle heat. The 
joint is wound with silk thread and coated with collodion. The sac is then filled 
with distilled water, immersed in a tube of water and sterilized in the autoclave. 

There are also various other methods recommended for making collodion sacs. 


Collodion capsules are ordinarily placed free in the peri- 
toneal cavity of the animal, by an aseptic laparotomy. The 
wound is sutured with silk or catgut and dressed with sterile cotton 
and collodion. 


1 Journal of Experimental Medicine. Vol. VI, p. 635. 
2 Contributions to Medical’Research. Dedicated to Victor C. Vaughan, Ann 
Arbor, 1903, p. 390. 


I40 BACTERIOLOGY 


Observation of Infected Animals—In nearly every case it will 
be well to keep a record of weight of the animal from time to time. 
The temperature may be observed by means of a thermometer 
in the rectum. It should be inserted a considerable distance, 
4 to 8 centimeters in guinea-pigs. Other examinations are made in 
special cases, such as palpation of the lymph glands in tubercu- 
losis and microscopic examination of the blood in anthrax, tryp- 
anosomiasis and the relapsing spirochetoses. . 

Post-Mortem Examination of experimental animals is often 
of great importance. The body is first soaked in bichloride solu- 
tion ‘to wet thoroughly the hair and skin. It is then fixed ona 
board by cords or by nails through the feet, stretched out with 
the ventral surface exposed. With sterile scissors an incision 
is carried through the skin in the median line from neck to pubis 
and branch incisions are carried to the extremities. The skin is 
reflected with aid of a scalpel and the desired examinations of the 
subcutaneous structures carried out. The abdominal muscle 
layer is then seared with a hot iron in the median line as are also 
the lateral walls of the thorax and with a new set of sterile in- 
struments the seared line is incised so as to expose completely 
the contents of abdomen and thorax. Heart’s blood is obtained 
by searing the epicardium and puncturing the right ventricle 
with a sterile glass Pasteur pipette. Any or all the organs of 
thorax and abdomen may then be removed to sterile glass dishes. 
Immediate microscopic examination for microorganisms is made 
by direct slide-coverglass preparations of the fresh material diluted 
with salt solution and by smearing the fluids and tissues on cover- 
glasses or slides and staining them by various methods. Cultures 
are also made and it is important to make plate cultures directly 
from the animal in all instances‘in which a mixed infection or 
possible contamination with extraneous organisms is suspected. 
To remove the spinal cord, the animal is turned so that the back 
is exposed, the skin divided by a median incision and stripped 
back to either side. The muscles are roughly dissected away from 
either side of the spinal column and the vertebral laminae are 


METHOD OF ANNUAL EXPERIMENTATION I4I 


broken through by bone-cutting forceps and the posterior sections 
of the vertebral arches removed from the lumbar region to the 
skull. The dural sheath of the cauda equina is firmly grasped by 
sterile forceps and the entire cord gradually lifted up as the spinal 
nerves are successively put on a stretch and divided by sterile 
scissors. Finally the cervical cord or the medulla is cut across 
and the cord placed in a sterile glass dish. In addition to the im- 
mediate examination it is well to place suitable pieces of tissue 
into fixing flujds for sectioning and histological study, by which the 
physical relations of the parasites and the tissue elements can 
be studied as well as the pathological alterations in the latter. 


PART II 


GENERAL BIOLOGY OF MICRO- 
ORGANISMS 


CHAPTER VII 
MORPHOLOGY AND CLASSIFICATION 


The minute living things included under the general term 
microbe, are exceedingly various in form and structure as well as 
in respect to food requirements and physiological activity. The 
number of different microbes is so great and so great are the diffi- 
culties involved in the accurate observation of their various 
features, that the biological relationships of many of the various 
forms to each other are not yet determined, and much of the 
generic and specific terminology in common use rests upon insecure 
foundation. Nevertheless a certain kind of order has developed 
in our conceptions of the grouping of micro-organisms. 

Molds and Yeasts.—The molds are multicellular organisms 
characterized by the formation of a network (mycelium) made up 
of branching threads (kyphe), and by their special fruiting organs. 
These threads vary from 2 to 7# in width. Within the group of 
molds the structure of fruiting organs is used as the most important 
character from which to determine relationships. The phycomy- 
cetes, or algo-fungi, are characterized by the presence of sexual 
reproduction in which the union of two cells gives rise to resting 
cells, zygospores and odspores, which are enclosed in a thick wall. 
The ascomycetes are characterized by a septate mycelium and 
by the occurrence of a spere-sac called the ascus, which usually 
contains eight spores but may contain a large number in some spe- 

143 


144 GENERAL BIOLOGY OF MICRO-ORGANISMS 


ETE 


By 


€ d € 
Fic. 61.—Asexual fruiting organs of common molds. 


a. Penicillium glaucum. 0b. Oidium lactis. c. Aspergillus glaucus. d. The same 
more highly magnified. e. Mucor mucedo. (Baumgarten.) 


MORPHOLOGY AND CLASSIFICATION 145 


cies. The common aspergilli belong here. The dbasidiomycetes 
are characterized by the occurrence of a spore-bearing cell, the 
basidium, which bears four protuberances called sterigmata 
(singular sterigma) upon each of which is a single spore. Mush- 
rooms and pufi-balls belong to this group. Besides these three 
well-defined classes, there are many kinds of molds and fungi con- 
cerning which definite knowledge is still too incomplete for them 
to be finally placed. These are designated as imperfect fungi, 


Fic. 62.—Yeast cells stained with fuchsin. ( X1000.) 


‘Fungi imperfecti, or perhaps best by the class name, Hypho- 
mycetes. In these forms, zygospores and ascospores are un- 
known; the hyphae are often septate. Reproduction takes place 
by the formation of conidia only. These are oval or rounded 
cells produced by transverse division of a filament, usually as a 
row of conidia at the end of a hypha. The common oidium and 
many parasitic molds belong in this class. The molds! are es- 
pecially important as causes of disease in plants. Relatively few 

1 For fuller discussion of molds in general see Marshall, Microbiology, article by 


Thom. 
10 


146 GENERAL BIOLOGY OF MICRO-ORGANISMS 


diseases of man or other animals have been shown to be due to 
them, although the first diseases proven to be due to micro-organ- 
isms were those caused by certain molds. The molds possess 
the general morphological features of plants except for the ab- 
sence of chlorophyll. 


Fic. 63.—Wine and beer yeasts. A. S. ellipsoideus, young and vigorous; B, S. 
ellipsoides, (1) old, (2) dead; C, S. cerevisie, bottom yeast; D, S. cerevisie, top yeast. 
(Original.) 

The yeasts, in general, are ovoid, specialized cells of molds, 
belonging to several different genera. The true yeasts, genus 
Saccharomyces, belong to the ascomycetes. They do not grow 
out into long filaments but remain spherical or ovoid. The cells 
vary from 2.5 to 124 in diameter. During active growth they 
reproduce by budding, a smaller portion being pinched off from the 
parent cell. The true yeasts also form spores inside the cell, 


MORPHOLOGY AND CLASSIFICATION 147 


from four to eight typical ascospores. Yeasts are very important 
in the fermentation industries. Very few of them are pathogenic. 
Among themselves, the yeasts are subdivided into two groups, 
(1) those which produce ascospores (saccharomycetes or true 
yeasts) and (2) those which fail to produce such spores (torule 
or wild yeasts). They are further distinguished by differences 
in the form of the cells, but especially by differences in physio- 
logical characters, such as the fermentation of sugars and the 
production of pigments. 

In the yeasts there is no definite differentiation of cells. Vari- 
ous cell structures such as cell-wall, nucleus and cytoplasm with 
vacuoles and granules, can be demonstrated. The cell membrane 
is, as a rule, more delicate than in the molds. It sometimes 
secretes a gelatinous material which forms a thick capsule about 
the cell. The nucleus is shown by appropriate methods of stain- 
ing as a single more or less sharply defined mass of chromatin. 
Under suitable conditions the true yeasts produce endospores, 
usually multiple, and as many as eight in one cell. These are 
spherical or ovoid masses surrounded by a definite wall, and usually 
about half the diameter of the yeast cell. When supplied with 
nutriment these spores swell and burst the mother cell, and then 
begin at once to multiply by budding. Dry commercial yeast 
cakes contain spores of yeast along with bacteria and molds; moist, 
“‘compressed,’’ yeast contains vegetating yeast cells, also mixed 
with other organisms. 

Bacteria.—Bacteria (schizomycetes) are minute unicellular 
organisms 0.2 to 4uin width, which multiply solely by simple trans- 
verse division (fission), ordinarily resulting in the production 
of two cells of equal size. In many instances the cells remain 
attached to each other so as to form long filaments. 

Trichobacteria.—Certain of them grow into long filaments 
without dividing at once into shorter segments. These forms 
which are classed as higher bacteria or trichobacteria, suggest 
a very close relationship to the molds and may, perhaps, be re- 
garded as intermediate between the molds and the lower bacteria. 


148 GENERAL BIOLOGY OF MICRO-ORGANISMS 


Many of them exhibit a differentiation of the filament into base 
and apex, some of them branch in an irregular fashion, and in 
some there is a suggestion of the formation of special fruit organs. 
These higher bacteria require further study to determine their 
relationships. A few of them are important pathogenic agents. 

The Lower Bacteria.—The lower bacteria, or true bacteria, 
are always simple in form, the transverse division producing 
cells, relatively short, and of nearly equal length. Long filaments 
are produced only by the attachment of many individual cells 
together, end to end. There are no special fruit organs. The 
special resistant form, or spore, which occurs in some forms, is 
produced only inside of the vegetative cell, one cell producing 
one spore. There are three general forms of bacteria, the sphere 
(coccus, plural cocci), the cylinder (bacillus, plural bacilli), and 
the spiral or segment of a spiral (spirillum, plural spirilla). In- 
termediate forms occur, so that-there is net a sharp line between 
the groups. These three forms are generally accepted as a basis 
for division of the lower bacteria into three families, the coccacezx, 
kacteriacee and the spirillacee. 

Spherical Bacteria~—The Coccacee cr cacci are spherical 
bacteria. They vary in size from about 0.34 to 3u in diameter. 


2 XG i & ge 


Staphylococci. Streptococci. Diplococci. Tetrads. Sarcine. 
Fic. 64. 
During the process of cell division, a coccus may become elongated 
somewhat, and after division, the daughter cells may be shortened 
so that they appear as if compressed against each other. Slightly 
elongated forms are included among the cocci in certain instances, 
and especially the lancet-shaped bacteria such as the germ of 
lobar pneumonia. The recognition of genera within the family 
is still unsettled. Morphologically five genera have been dis- 
tinguished by Migula: Streptococcus, Micrococcus, Sarcina, 


MORPHOLOGY AND CLASSIFICATION 149 


Planococcus and Planosarcina. The first three do not possess 
flagella and are non-motile. Streptococcus includes those forms 
which divide only in one plane so that a thread or chain is produced. 
Micrococcus includes the cocci which divide in two planes at 
right angles so as to produce plates, and it also includes those 
which divide in an irregular fashion so that no definite geometric 
figure results. Sarcina includes those cocci which divide in three 
planes at right angles to each other, in turn, so as to produce 
cubical masses of cells. Planococcus is similar to Micrococcus 
in all respects except that its members are motile and possess 
flagella, and Planosarcina includes the motile forms which are 
in other respects the same as the forms included under Sarcina. 
COCCACEA—Cells spherical, without endospores. 

Streptococcus—Division in one plane, forming chains of cells; 
non-motile; without flagella. 

Micrococcus—Division in two planes, forming flat plates of 
cells, or irregular, forming masses of cells irregularly 
grouped; non-motile; withcut flagella. 

Sarcina—Division in three planes, forming cubical or package- 
shaped masses cf cells; non-motile; without flagella. 

Planococcus—Division in two planes, forming flat plates 
of cells, or irregular, forming mass of cells irregularly 
grouped; motile; bear flagella. 

Planosarcina—Division in three planes, forming cubical 

-cr package-shaped masses of cells; motile; bear flagella. 
These genera have not been generally adopted by bacteriolo- 
gists. The terms Streptococcus and Sarcina are, however, 
quite generally employed as the generic names for the organisms 
of their respective groups as defined by Migula, as they had 
been used in this way before. Micrococcus, however, is commonly 
employed as a general term for all the members of the family 
Coccacez, and Planococcus and Planosarcina have not been used, 
because bacterial forms belonging to these genera are exceedingly 
uncommon and it may even be questioned whether those which 
have been described might not better be classed with the cylin- 


NS 


150 GENERAL BIOLOGY OF MICRO-ORGANISMS 


drical bacteria, in which motility is of frequent occurrence. 
Other terms in common use as generic names for certain cocci 
are Diplococcus and Staphylococcus. A diplococcus is a double 
coccus, two spheres attached together. This grouping by twos 
is very common and the generic term Diplococcus is employed 
for those forms in which it is a prominent characteristic. The 
term Staphyloccccus is applied to those micrococci which are 
grouped in an irregular mass resembling a bunch of grapes. 

Cylindrical Bacteria.—The cylindrical bacteria, Bacteriacee, 
have been subdivided by Migula into three genera, Bacterium, 
Bacillus and Pseudomonas. The genus Bacterium includes 
those members of the family which are without flagella and 
are non-motile. Bacillus includes those forms possessing flagella 
distributed over the surface, and Pseudomonas is the generic 
term for those forms with flagella situated at the extremities 
only (polar flagella). 

BACTERIACEA—Cells cylindrical, straight, non-motile 
or motile by means of flagella. 
Bacterium—Cells without flagella, non-motile. 
Bacillus—Cells motile with flagella distributed over the 
surface. 
Pseudomonas—Cells motile with polar flagella. 

These genera have not been generally adopted by bacteri- 
ologists, and there are serious reasons for dissatisfaction with 
such a classification of the rod-shaped bacteria. In the first 
place the names Bacterium and Bacillus are unfortunate. The 
former has long been employed as a general term designating 
any member of the Schizomycetes and its plural, Bacteria, 
is everywhere the common term employed in designating this 
large group of micro-organisms. Its use in the narrower sense 
by Migula has not displaced the former signification, and its 
use in the sense of Migula must necessarily result in confusion. 
The latter term, Bacillus, has long been used very generally by 
bacteriologists to designate any member of the Bacteriacer 
or rod-shaped bacteria, regardless of the motility or distribution 


MORPHOLOGY AND CLASSIFICATION I5i 


of flagella. A further serious objection is due to the lack of 
stability in the character selected to distinguish the genera. 
The flagella may disappear from bacteria ordinarily possessing 
them as a result of changes in environment and may be again 
made to appear by reversing the conditions.! Furthermore 
in some groups of bacteria, which seem to be closely related in 
respect to other characters, morphological and physiological, 


we aU Y 


Fic. 65.—Bacilli of various forms. 


4 
both motile and non-motile forms occur.- On the whole the pres- 
- ence or absence of flagella would seem to be too fragile a character 
to serve as a sole distinction between genera among the rod- 
shaped bacteria. 

The different species of rod-shaped bacteria are very numerous, 
several thousand different kinds having been described. They 
vary in width from 4u to o.1p or probably less, and in length.frem 
6ou to 0.24. The very large ones are non-pathogenic species. 


Gee) CB Ce 


Fic. 66.—Sporulation. a, First stage showing sporogenic granules; b, incomplete 
spore; c, fully developed spore. (After Novy.) 


The form is ordinarily that of a straight cylinder of equal caliber 
throughout its length. Certain slightly curved forms are never- 
theless included in the family, although they may perhaps be 
regarded as intermediate between the bacteriacee and the 
spirillacee. Some of the rod-shaped bacteria are of uneven 
caliber, especially when growing under unfavorable conditions or 
when spores are produced. The ends of the rod may be pointed, 
rounded, square-cut or concave. The bacteria may remain at- 


1Passini: Zis. f. Hyg., 1905, XLIX, pp. 135-160. 


152 GENERAL BIOLOGY OF MICRO-ORGANISMS 


tached after cell-division, forming groups of two, dzplo-bacillus, 
or many cells remain attached, to form long threads, strepto- 
bacillus. Endospore formaticn occurs almost exclusively in 
the bacteriacee and the form of the spore-bearing cell differs 
for different species and is fairly constant for any one species. 


yy 


SSSSKC“S 


Fic. 67.—Position of spores; resultant forms (diagrammatic). a, Median 
spores; b, intermediate spores; c, terminal spores; 2a, b, c, change in form of cells 
due to the presence of the sp ore; 2a, clostridium; 2c, drum-stick form. (After Novy.) 


The spore, which is always single, may be located at the center of 
the cell, median spore, or at the end, terminal spore, or at an 
intermediate point. The spore-bearing cell may retain its normal 
outline or it may be bulged by the spore. The cell containing 
a median spore with bulging is called a clostridium; one with 
terminal spore with enlargement of the cell is spoken of as a drum- 
stick or sometimes as a plectridium. 

Spiral Bacteria.—The screw-shaped bacteria, Spirillacee, have 
been subdivided into four genera by Migula. The genus Spiro- 
soma includes those spirals which are rigid and without motility. 
Mcotile cells possessing one, two or three polar flagella are classed 
in the genus Microspira; while those possessing more than three 
are put in the genus Spirillum. The genus Spirocheta includes 
the slender flexuous forms of spirals. 

SPIRILLACEA—Cells circular in cross-section but 
curved to form a spiral or segment of a spiral. 

Spirosoma—Cells rigid, without flagella, motionless. 

Microspira—Cells rigid, motile, with 1 to 3 polar flagella. 

Spirillum—Cells rigid, motile, with polar tufts of flagella. 

Spirocheta—Cells slender, flexuous, motile. 


MORPHOLOGY AND CLASSIFICATION (153 


Two of these generic terms, Spirillum and Spirocheta, have 
long been used, and almost in the sense in which they are em- 
ployed by Migula. Spirillum has frequently been applied to 
all the Spirillaceze and especially to those forms which Migula 
includes in his first three genera, Spirosoma, Microspira and 
Spirillum. The distinction between Microspora and Spirillum 
seems of too slight importance to serve as a basis for the formation 
of two gerera, and indeed 


the same objection exists / Pod 
here as in the Bacteriacee hes Os aye ¥ chs 
to the use of flagella as F ~~ 


" 
a basis for generic dis- (nee 


tinctions. Fic. 68.—Types of spirilla. 
Cell division occurs by 


simple transverse fission in all the spiral bacteria. Endospores are 
said to be formed by some species. 

The group of spirochetes has. received much attention in 
recent years and the propriety of including them in the spirillacez 
may be seriously questioned. Many investigators are inclined 
to regard them as more properly classed with the protozoa than 
with the bacteria. It is claimed that these forms multiply by 
longitudinal splitting and not by transverse fission, and this would 
at once remove them from the Schizomycetes. The observations 
are still in dispute and there are good observers who regard trans- 
verse fission as the mode of multiplication. Further study is 
necessary to settle this important question. Itis possible that some 
of these slender spirals may multiply by both methods, or that 
one species may divide longitudinally and another transversély, 
but this does not seem probable. For the present it would seem 
wise to reserve judgment and avoid encumbering the group with 
new genera until a definite and final agreement has been reached 
concerning the exact morphological facts. (See page 368.) 

Structure of Lower Bacteria.—The bacterial cell is enclosed 
in a relatively stiff cell membrane, which generally retains its form 
after plasmolysis. Under special conditions of growth many 


wt 


154 GENERAL BIOLOGY OF MICRO-ORGANISMS 


forms of bacteria become enclosed in a gelatinous capsule. This 
seems to be a viscid material secreted by the cell through the cell 
membrane. The motile bacteria possess exceedingly slender hair- 
like processes, called flagella, which serve as organs of lecomotion. 
These processes apparently take origin from the cell membrane. 
Bacteria without flagella are spoken of as 

© ® t atrichous, those with a single flagellum at 
0@ 4} bn & @ one end as monotrichous, those with a flag- 
fy ellum at either end asamphitrichous. When 

© 0 == there is a tuft of flagella at the end, the dis- 
Bias gi with tribution is said to be lophotrichous, and 
when they are distributed all over the sur- 

face the arrangement is called peritrichous. The internal 
structure of the bacterial cell has received comparatively little 
attention. The direct microscopic study of the living cells 
shows them:to be finely or coarsely granular, or sometimes nearly 
homogeneous. No constant internal structures can be distin- 
guished. Ordinary simple staining with the kasic aniline dyes 
colors the bacterial cell diffusely and intensely, usually with- 
out any internal diterenintiien, The cell membrane between 


Ids Se NK #& 


Fic. 70.—Bacteria showing flagella. 


two cells in a chain may remain relatively colorless Ha so be 
differentiated from the protoplasm on either side. At times the 
stainable substance is unevenly distributed in the cell, perhaps 
grouped at the ends of a rod, or in granules or bands. Under 
special conditions some bacteria show internal granules of special 
composition, distinguishable as pigment granules or by their 
microchemical reactions. Granules which stain with iodine, so- 
called granulose or glycogen granules, are important features 
of some kinds of bacteria. 


MORPHOLOGY AND CLASSIFICATION 155 


The recognition of the cell nucleus has received special atten- 
tion. Zettnow, more especially, has shown that the chromatin or 
essential nuclear substance is present in 
the bacterial cell as finer or larger granules, 
sometimes distributed pretty generally 
and sometimes collected together at one 
or more places in the.cell. The Roman- 
owsky stain and its modifications have 


é ‘ ae ae Fic. 71.—The formation 
been especially useful in differentiation of of spores. (After Fischer 


chromatin from cyt opl asm from Frost and McCampbell.) 


Special movements of the internal granules have been described 
by Schaudinn as being associated with beginning cell division. 
For the great majority of bacteria these have not been observed, 


oe ef ) g J 
‘aay Ui. x S 


Fic. 72.—Bacteria with spores. 


and according to our knowledge, the process of cell division is ex- 
tremely simple. It consists of a progressive constriction and thin- 
ning of the cell at the middle until two cells are produced. In 


ee @2o > 00 C O= (= Qa 


ooh) a ; 


Fic. 73.—Germination of spores. a, Direct conversion of a spore into a bacillus 
without the shedding of a spore-wall (B. leptosporus); b, polar. germination of B. 
anthracis, c, equatorial germination of B. subtilis; d, same of B. megatherium; e, 
same with “‘horse-shoe” presentation. (After Novy.) 


some forms the division is completed by a sudden snapping move- 
ment. ; 


156 GENERAL BIOLOGY OF MICRO-ORGANISMS 


The formation of an endospore begins with the accumulation 
of chromatin granules in one part of the cell, where they coalesce. 
lose their contained water and seem to become embedded in an oily 
or fatty substance and surrounded by a membrane. Very early in 
the process the spore no longer stains readily. In some forms 
(Bact. anthracis) the cell in which a spore has formed disintegrates 
rapidly, setting free the spore, while in others (B. tetani) the cell 
may continue its activities after formation of the spore. The spore 
‘germinates when conditions again become favorable to active 
growth. The new cell may burst the spore wall into halves, or 
at the end, or the spore wall may soften and become a part of the 
new growing cell. 

Filterable Viruses.—The difficulty of accurate morphological 
study is so great as to appear insurmountable in the case of cer- 
tain microbes which are very definitely recognizable by certain 
effects which they produce. This is especially true of those living 
things capable of passing through the fine filters which prevent 
the passage of small bacteria. The causes of certain diseases 
exhibit this character, and these have come to be known as filter- 
able viruses. There can ke little question that non-pathogenic 
filterable microbes also exist although they seem to have escaped 
observation. Accurate knowledge of the morphology of many 
of these forms remains to be disclosed by future investigation. 
Meanwhile, the efforts to classify them as bacteria or as protozoa 
may well be spared. The propriety of including them as living 
things is, however, only occasionally questioned. 

Protozoa.—The prctozoa or unicellular animals have assumed 
very great importance as causes of disease during the past twenty 
years. Fortunately for the systematist, the free-living protozoa 
had received considerable careful study and the larger groups of 
protozoa had been well defined before the interest in pathogenic 
properties had the opportunity to over-shadow morphological 
study. The number and variety of easily recognizable morpho- 
logical characters presented by the protozoa are greater than 
those of the bacteria; and the organisms are, on the whole, larger. 


MORPHOLOGY AND CLASSIFICATION 157 


These factors make for more accurate observations of morpho- 
logical characters, and their more successful employment as a 
basis of classification. 

The protozoan cell is generally larger and more complex in 
structure than the bacterial cell appears to be, although the di- 
viding line is in places indefinite or even wholly obscure. In 
general the protozoon shows the typical structure of a single cell - 
of the metazoon. A well-defined nucleus is usually present, some- 
times several of them, although in some forms the nuclear ma- 
terial is more or less scattered throughout the cell. Most protozoa 
exhibit differentiation of the protoplasm into cell organs or 
organelle, adapted to perform certain functions. In many pro- 
tozoa sexual reproduction has been observed, a process involving 
complex morphological changes. The cells showing these evi- 
dences of complex organization resemble in most respects cells 
of the higher animals, and in fact a colony or group of protozoa 
may be regarded as representing a transition to the many-celled 
animals, just as, on the other hand, the bacteria were seen to be 
connected with the higher plants through the forms of the higher 

bacteria, the yeasts, the molds and alge. Physiologically, pro- 

tozoa differ from bacteria and other plants in requiring more com- 
plex nitrogenous food, but this distinction is far from absolute. 
Doflein divides the protozoa into two substems, (1) Plasmodroma, 
including those forms which move by means of pseudopodia or 
flagella, and which exhibit for the most part an alternation of 
asexual and sexual generations, and (2) Ciliophora, including 
those forms which move by means of cilia and in which the sexual 
fertilization gives rise to no special reproductive form of the 
organism. 

The substem Plasmodroma includes three classes, (1) Masti- 
gophora, (2) Rhizopoda and (3) Sporozoa. 

Flagellates——In the class Mastigophora, are included a great 
many different organisms, the one common feature being the 
type of locomotive apparatus, which consists of cne or more flagella. 
The further subdivision of the class has not yet been agreed 


158 GENERAL BIOLOGY OF MICRO-ORGANISMS 


Fic. 74.—The most important trypanosomes parasitic in mammals. A, Try- 
panosoma lewisi (Kent).- B, Tr. evansi (Steele), Indian variety. C, Tr. evansi 
(Steele), Mauritian variety. D, Tr. brucei (Plimmer and Bradford). £, Tr. equip- 
erdum (Doflein). F, Tr. equinum (Voges). G, Tr. dimorphon (Laveran and Mesnil), 
H, Tr. gambiense (Dutton). (From Doflein after photomicrographs of Novy.) 


Fic. 75.—Leishmania donovani. Various forms obtained by spleen puncture, some 
free and some inside red blood cells. (From Doflein after Donovan.) 


MORPHOLOGY AND CLASSIFICATION 159 


\ 
upon, not because of any lack of morphological differences upon 
which to base a classification, but largely on account of difficulty 
in estimating the relative importance and meaning of the many 


( A 
Fic. 76.—Leishmania Fic. 77.—Trichomonas hominis. 
donovani. Various forms (From Doflein after Grassi.) 
of the organism in artificial 


culture. (From Doflein after 
Chatterjee.) 


criteria presented. The genera of particular interest from the 
pathological standpoint are Trypanosoma, Leishmania, Tricho- 


A B C 
Fic. 78.—Lamblia intestinalis. A, Ventral aspect. B, Lateral aspect. C, At- 
tached to an epithelial cell. (From Doflein after Grassi and Schewiakoff.) 


monas and Lamblia. The members of the Trypanosomata are 
characterized by an approximately crescent-shaped body, 10 to 
4ou in length, flexible and provided with a flagellum which origi- 


160 GENERAL BIOLOGY OF MICRO-ORGANISMS 


nates in the endoplasm near one end and passes along the border 
of the body and finally projects as a free whip at the other end 
of the cell. As it passes along the border of the cell it is enclosed 
in a sheath of ectoplasm, which is drawn out into a thin sheet 
forming an undulating membrane. Multiplication takes place 
by approximately longitudinal division. Leishmania includes a 
few parasitic forms, for the most part living inside the cells of 
the host. These organisms are oval, about 2X3u, without fla- 


Fic. 79.—Endameba coli (Lésch). A to C, Various forms of the free ameba. 
D, Stage with eight nuclei. E to G, Cysts with various numbers of nuclei. 4H, 
Opening cyst. J, Young amebe escaped from a cyst. (From Doflein after Casa- 
grandi and Barbagallo.) 


gellum or undulating membrane. In artificial culture outside 
the body, the protozoon grows larger, develops a flagellum and 
resembles a trypanosome. Trichomonas includes pear-shaped 
organisms 4 to 30m in diameter, provided with three or four fla-. 
gella. Isogamic and autogamic fertilization have been described, 
and cysts containing numerous daughter cells result from the 
multiplication following this process. Lamblia resembles tricho- 
monas, but the cell is here shaped more like a beet, is provided 


MORPHOLOGY AND CLASSIFICATION 161 


with eight flagella and is hollowed out at one side near the rounded 
anterior end to form a suction cavity. 

Rhizopods.—The members of the second class, Rhizopoda, 
are characterized by their ability to send out protoplasmic proc- 
esses to serve for locomotion and also to surround and engulf 
solid food particles. The two genera, Ameba and Endameba, 
are of chiefest interest. The organisms are masses of protoplasm 
containing a nucleus, food granules and sometimes vacuoles, and 
surrounded by a slightly denser more hyaline layer of ectoplasm. 
The members of the genus Ameba are free-living saprophytic 
forms, while those of Endameba are parasitic. Multiplication 
occurs by fission after a more or less complex division of the 
nucleus. Multiple division also occurs, more especially in an 
encysted condition, and- subsequent to a possible autogamic 
fertilization. 

Sporozoa.—The third class, Sporozoa, is made up entirely 
of parasitic forms, which at some stage in their life history multiply 
by division into numerous daughter cells, which are enclosed in a 
protective envelope to form a spore. The spores serve to dis- 
tribute the species to other hosts. In cases where there are special 
adaptations for distribution, as for example by means of inter- 
mediate hosts, the protective envelope may be absent. An enor- 
mous number of parasitic micro-organisms are included in this 
group. The genera of greatest present interest from the patho- 
logical point of view are Eimeria (Coccidium), Plasmodium, 
Babesia (Piroplasma) and Nosema. 

The Coccidia—Eimeria includes a number of. intracellular 
parasitic forms, perhaps better known as coccidia. The small 
parasite resulting from asexual division is called a merozoit. It 
is somewhat spindle-shaped and 5 to tou long. This merozoit 
- penetrates an epithelial cell of the host, grows at the expense of 
the cell to a spherical mass 20 to sou in diameter, and eventually 
divides into numerous (sometimes as many as 200) merozoits, 
which become free by rupture of the host cell. Besides this asex- 


ual mode of multiplication, there is also a sexual cycle. Some of 
7 


162 GENERAL BIOLOGY OF MICRO-ORGANISMS 


‘ 


Fic. 80.—Developmental cycle of Eimeria (Coccidium) schubergi. I, Sporozoit; 
II, sporozoit penetrating a cell of the host; IJI and IV, stages of growth; V to 
VII, asexual multiplication; VIII, agamete or merozoit beginning again the asexual 
cycle; IX and X, agametes destined to form sexual cells (gametes) ; XI, a to c, devel- 
opment of the macrogamete; XII, a to d, development of microgametes; XIII, 
fertilization; XIV and XV, the fertilized cell or zygote; XVI and XVII, metagamic 
division of the zygote; XVIII, formation of the sporoblasts; XIX, formation of 
the spores and sporozoits; XX, sporozoits emerging from the spores and from the 
oocyst. (From Doflein after Schaudinn.) 


MORPHOLOGY AND CLASSIFICATION 163 


the growing parasites do not divide into merozoits but become 
differentiated into male and female cells (gametocytes). The 
male gametocyte gives rise to a large number of elongated motile 
microgametes, one of which approaches and penetrates the 
ripened macrogamete. The nuclei of the two gametes fuse and 
the fertilized cell quickly forms a protective wall around itself and 
then divides into eight cells which are enclosed in pairs within 
secondary cysts known as spores. This form of the organism 
passes out of the host, and after a passive existence in the external 


Fic. 81.—Forms in the asexual cycle of Plasmodium falciparum, the parasite 
of tropical malaria. A, Multiple infection of a red blood cell; B to EZ, various forms 
of the growing parasite; B and C show also the Maurer granulations; F, full-grown 
parasite with many nuclei; G, Segmentation. The pigment is shown in FE, F andG. 
(After Doflein.) 


world may gain entrance to a new host, whereupon the spore wall 
ruptures and the enclosed cells, sporozoits, emerge to penetrate 
new host cells. 

The Plasmodia.—Plasmodium includes the malarial parasites, 
forms parasitic in red blood cells and closely analogous to the 
coccidia in the asexual cycle. The gametocytes are also similar to 
those of Eimeria except that the gametes are not formed within 
the mammalian host, but only after the blood has been drawn. 
The sexual cycle of development takes place in a definite secondary 


164 GENERAL BIOLOGY OF MICRO-ORUVANISMS 


host, the mosquito. In the stomach of this insect the gametes 
unite and the fertilized cell (odkinet): actively penetrates the 
epithelium and beneath it develops into a large odcyst, 30 to gou 
in diameter, enclosed in the elastic tunic of the stomach wall of the 
mosquito. As the odcyst enlarges, the nucleus divides and eventu-- 
ally the cytoplasm also. The nucleus of each of these masses 
(sporoblasts) then divides many times. Each nucleus, together 
with a small amount of protoplasm, separates and then elongates 
into a slender thread-like sporozoit (14 X 1m). AS many as 10,000 


Fic. 82.—Babesia muris. A, Young form in a red blood cell. B, Form with 
two nuclei. C and D, Binary division. E and F, Multiple infection; ameboid 
forms in F. G, An exceptionally large individual (gametocyte?). H, Form with a 
thread-like process (flagellated stage?). (From Doflein after Fantham.) 


of these may be produced in one odcyst. The cyst bursts into the 
body cavity of the mosquito and the motile sporozoits circulate 
through the body of the insect and eventually assemble in the cells 
of the salivary glands. From these they escape with the secretion 
and gain entrance to the wound made by the mosquito in biting. 

Babesia.—A number of parasites of the red blood cells are 
classed in the genus Babesia (Piroplasma). These resemble the 
members of the preceding genus very closely but multiple division 
(segmentation) does not seem to occur in the asexual cycle. The 
multiplication seems to be by longitudinal division into two 
daughter cells. The characteristic form is pear-shaped, but 
irregular amceboid forms are also common. Flagellate stages 
existing in the blood plasma have also been described. The sexual 


MORPHOLOGY AND CLASSIFICATION 16 5 


cycle takes place in a tick, and is in part analogous to that de- 
scribed for Plasmodium. The stages are not fully known, but the 
infectivity of the tick is transmitted to the offspring in the case of 
the Texas-fever tick (Rhipicephalus (Boophilus) annulatus). 
Nosema.—The sporozoa above described all belong to the 
Telosporidia, organisms which end their individual existence at 


pa fe 
ass 


Fic. 83.—Diagram of the developmental cycle of Nosema bombycis. C, Cell of 
the intestinal epithelium containing asexual multiplication forms and showing their 
transition into spores. u, b, c, Spores, the last with polar thread. d, Ameboid form 
emerging from the spore to penetrate a new host cell at h. (From Doflein after 
Stem pell.) 
the stage of spore formation. A second large subdivision of the 
sporozoa is named Neosporidia. In this group the spores are 
formed without terminating the existence of the individual. The 
parasites of this type are comparatively small and not very well 
known. Theyareoften spoken of as microsporidia or psorosperms. 
The best-known form is Nosema bombycis, the cause of Pébrine in 


silkworms. 


166 


GENERAL BIOLOGY OF MICRO-ORGANISMS 


Ciliates.—The' second substem of the protozoa, ‘Ciliophora, 
is distinguished by the locomotive organs, numerous cilia which 
cover most of the body surface, and by the possession of two dis- 
tinctly different nuclei, one apparently concerned with nutrition 
of the cell and the other definitely associated in an important man- 


ner with the sexual reproduction. 


Multiplication takes place by 


transverse division into two daughter cells or by budding. In the 
parasitic forms this may take place within a protecting wall (cyst). 
The sexual fertilization is‘not followed by any special kind of 
division. Balantidium is the only genus of present interest as a 
cause of human disease. See Balantidium coli, p. 454. . 


Protista 
(Microbes) 


OUTLINE CLASSIFICATION OF Micro-oRGANISMS 


Fungi 
(Plants) 


Not classified—Filterable microbes 


Protozoa 


(Animals) 


Phycomycetes 


Ascomycetes 


Hyphomycetes 
Fungi imperfecti 


Schizomycetes 
(Bacteria) 


Plasmodroma 


Ciliophora 


Rhizopus 
Zygomycetes ; Mucor 
‘| Thamnidium 
Oémycetes—Mildews 


Aspergillus 


_| Penicillium 


Claviceps 
Saccharomyces. 
Coccidioides - 


Oidium 
Monilia 
Botrytis 
Sporotrichum 
Cryptococcus 


Trichobacteriaceae 
Coccaceae 
Bateriaceae 
Spirillaceae 


Mastigophora 
Rhizopoda 
Sporozoa 


Ciliata 
Suctoria 


MORPHOLOGY AND CLASSIFICATION “167 


Classification and Specific Nomenclature.—The classification 
of microbes is at present in an unsatisfactory state. In part this 
situation may be ascribed to the difficulty of ascertaining and 
observing accurately the features important for classification in 
this group of living things. In part, however, the confusion 
depends upon a too little controlled activity in the creation of 
new names by authors unwilling to expend the time and labor to 
become familiar with the old names. One should not lightly 
create new genera and the author who creates a new genus may 
well be called upon to prove the necessity for its creation before 
the added burden is tacked on to our nomenclature. Generic 
names are subject to revision as are also the names of families and 
larger groups. It is hoped that some authoritative body such as 
an international committee of bacteriologists or of general biolo- 
gists, will in the near future decide upon a definite scheme for 
the classification of the fungi and especially the schizomycetes. 
A committee of the Society of American Bacteriologists has pub- 
lished’ a proposed classification which may serve to further an 
international agreement. At present the introduction of such a 
more elaborate and still somewhat unsettled classification into 
an elementary textbook, would seem premature. 

The classification of the protozoa is in a more satisfactory state, 
largely because of the monumental work of Doflein? but even in 
this realm the careless creation of new genera and the use of differ- 
ent generic names for the same organism by different authors is 
to. be regretted. 

A species is properly designated by a Latin binomial, the first 
member of the name being the name of the genus and the second 
member the specific name, such, for example, as Mucor mucedo, 
Saccharomyces cerevisiae, Bacillus coli, Spirochaeta pallida, Plas- 
modium falciparum and Balantidium coli. Bacillus is the generic 
term and coli the specific term. A third term is allowable to 


1 Winslow, Broadhurst, Buchanan, Krumwiede, Rogers and Smith: The families 
and genera of the bacteria, Journal of Bacteriology, 1917, 2, Pp. 505. 
? Doflein, F., Lehrbuch der Protozoenkunde, Jena, 1911. 


168 GENERAL BIOLOGY OF MICRO-ORGANISMS 


designate a variety of a species, such use being only. temporary 
until a decision can be reached as to the relationship of the new 
organism under consideration, as for example Bacillus colt com- 
munior. Subsequently if the new organism proves to be Bacillus 
coli, the variety name communior may. be dropped. On the other 
hand if it proves to be distinct from Bacillus coli the old variety 
name should then become the specific name resulting in Bacillus 
communior. The specific name is a single and very definite term 
and as a rule it is either the first published name given tothe organ- 
ism or some emended adaptation of it, in proper grammatical 
agreement with the generic term employed. Thus in designating 
the parasite of syphilis; one may employ the term, Spirochaeta 
pallida classing it in the genus Spirochaeta (Ehrenberg), but if 
the proposed genus Treponema (Schaudinn) be adopted, the name 
becomes Treponema pallidum. . 


CHAPTER VIII 
PHYSIOLOGY OF MICRO-ORGANISMS 


Relations of Morphology and Physiology.—In morpholegica] 
study observations are restricted to the relationship of various 
elements at a given time, facts relating to form and structure. 
From the physiological viewpoint one is more interested in the 
sequence of events and the relation of cause and effect. The 
possible suggestion that these two methods of study are independ- 
ent or mutually exclusive would be most unfortunate and is really 
very fallacious. The sequence of events may often best be ascer- 
tained by a series of morphological observations of a microbe 
undergoing change of form, and certainly the form and structure 
of a living organism at a given time may be properly regarded as 

an expression and result of previous physiological activity as well 
' the most essential element in its potentiality for future activity. 
All must agree that difference in behavior, that is, reaction to a 
definite environmental change, is really associated with a difference 
in structure of the living organism. The important difficulty 
lies in the fact that ‘the morphological or structural difference 
with which this difference in reaction is correlated, may not be 
capable of direct observation by any known method and may be 
ascertainable only by means of the physiological test. On the 
other hand the method of experimental physiology involves the 
factor of environment, small and unmeasured differences in which 
may grossly influence the resulting phenomencn and lead to erro- 
neous conclusions. Furthermore, the experimental conditions and 
the method of physiological observations may be wholly lacking 
in adaptation to potentialities of the organisms under observation. 
When properly employed, however, the method of experimental 


physiology yields valuable knowledge obtainable in no other way, 
169 


170 GENERAL BIOLOGY OF MICRO-ORGANISMS 


and it has been the most important single method in establishing 
our modern ideas of the relation of micro-organisms to infectious 
diseases, and is the method of greatest promise for the immediate 
future. 

Conditions of Physiological Study.—The physiology of many 
organisms is subject to only very limited experimental investi- 
gation. Those organisms of very narrow biological adaptation, 
such as many of the parasitic protozoa, can be studied only in 
very close relation to their natural environment, the. various 
important elements of which are not readily subject to experi- 
mental alteration and are largely unrecognizable. Our knowl- 
edge of these forms must therefore be derived almost exclusively 
from observations of form and structure, physical and chemical, as 
they exist and change under the natural conditions of environment, 
and from changes which take place in the tissues surrounding 
the parasite, which we may ascribe with more or less justifica- 
tion to their activity. Practically all that we know about the 
physiological activity of the very numerous microbes not yet 
brought into the group of artificially cultivable forms, has been 
deduced from morphological observations. Even observations 
of this kind, however, can be more successfully pursued in those 
forms capable of artificial culture, and artificial culture is a prime 
necessity for the study of cause and effect by the methods of ex- 
perimental physiology. For this reason accurate knowledge of 
what micro-organisms do is much richer in regard to the cultivable 
forms such as bacteria, yeasts and molds. In fact the microbic 
pure culture presents the most favorable object known for the 
study of cellular physiology and bio-chemistry. Furthermore, 
the physiological activities of many microbes are of the greatest 
practical importance. It is not surprising, therefore, that, 
among the bacteria, many of which grow in artificial media under 
a great variety of environmental conditions, the relative ease of 
physiological experimentation, as compared with the difficulty of 
observation of the minute morphological details, and the great 
practical importance of the results of physiological study has lead 


PHYSIOLOGY OF MICRO-ORGANISMS I7I° 


to an enormous development of knowledge gained by this method, 
which quite over-shadows our knowledge of morphology and 
structure in this group of organisms. 


THE INFLUENCE OF ENVIRONMENTAL FACTORS 


Moisture.— Moisture is indispensable to the growth of mi- 
cro-organisms. A few species will grow and multiply in almost 
pure distilled water. Drying causes the death of the majority of 
the vegetating cells, of some more readily than others, while the 
spore forms may remain alive in a dry condition for many: years. 
The cholera germ is dead within half an hour after it has been 
dried on a coverglass; the typhoid bacillus sutvives drying for 
days and the tubercle bacillus fcr months. Spores of the anthrax 
bacillus survive in the dry state for years, and perhaps indefinitely. . 

Heim! found that pathogenic bacteria resist drying much 
longer when contained in pathological tissues or exudates from 
animals which have succumbed to the disease, than when they 
are taken from artificial cultures. 

Organic Food.—One species of bacteria, Nitrosomonas cf 
Winogradsky, lives, grows and multiplies without organic food, 
utilizing the gases of the atmosphere as its scurce of carbon and 
nitrogen. From the standpoint of nutrition this organism is 
among the most primitive of beings. Other bacteria are known 
which may grow in water containing only mineral salts and a 
simple sugar, utilizing large quantities of atmospheric nitrogen. 
These are known as nitrogen-fixing bacteria. Most of the bac- 
teria, yeasts and molds require a small amount of nitrogenous 
organic matter as focd, such as the amino-acids or albumoses, and 
many of them flourish better when furnished a fermentable 
carbohydrate such as dextrose. The complex organic molecules 
are utilized in part to build up the substance of the bacteria, but 
a much larger part of them is broken down into simpler and more 
stable substances, such as carbon dioxide, simple fatty acids, 
ammonia and water, with the liberation of energy. Sapro- 


1 Zeitschrift f. Hygiene, Apr. 4, 1905, Bd. L, No. 1, p. 123. 


172 " GENERAL BIOLOGY OF MICRO-ORGANISMS — 


phytic organisms are those which grow on dead organic matter. 
Micro-organisms of still narrower adaptibility grow well in artifi- 
cial culture only if they be furnished abundant protein or nucleo- 
protein. Some important disease-producing bacteria Lelong in 
this category, as well as many parasitic spirochetes and some of 
the protozoa. Such organisms are not adapted to any natural 
saprophytic existence, and they grow in the artificial cultures 
only because the dead medium is made to resemble somewhat 
their natural parasitic habitat. Finally there are the. micro-or- 
ganisms which have not yet been grown in artificial culture and 
whose food requirements are essentially unknown. Many of 
these are parasites, and are called obligate parasites. A few bac- 
eria, many of the filterable agents, and most of the parasitic 
protozoa are included in this category. 

Inorganic Salts and Chemical Reaction.—Phosphorus, sul- 
phur, chlorine, calcium, sodium and potassium, in additicn to 
carbon, hydrogen, oxygen and nitrogen, are present as constituents 
of the microbic protoplasm. Minute quantities of these suffice 
to supply the focd requirements of micro-organisms and it is 
unnecessary to add them to culture media to serve as food. Com- 
mon salt, sodium chloride, is ordinarily employed to give the 
artificial medium an osmotic tension approaching that of the 
body fluids, and calcium carbonate is sometimes used to neutral- 
ize the organic acids which may arise in the culture as a result of 
the bacterial growth. . 

The most favorable chemical reaction for most micro-organisms 
is that of actual slight alkalinity, not sufficiently alkaline to pro- 
duce a red color with phenolphthalein and not sufficiently acid 
tc produce a red color with litmus. Some bacteria and many of 
the yeasts and molds will grow well in a weakly acid medium, 
but most parasitic bacteria and protozoa, which can be cultivated 
at all, require a reaction slightly alkaline to litmus or rosolic 
acid. The anaérobic bacteria do best in a medium containing 
glucose and with a reaction quite alkaline, indeed very close to 
the point at which phenolphthalein becomes pink. Organisms 


PHYSIOLOGY OF MICRO-ORGANISMS 173 


which produce acid or alkali are usually arrested in their growth as 
soon as a certain concentration is reached, and the medium may 
then rapidly kill the micro-organisms. 

Oxygen.—Oxygen, either free as atmospheric oxygen or com- 
bined as in water or organic compounds, is an essential constitu- 
ent of the food of all micro-organisms. The concentration of 
uncombined oxygen dissolved in the medium, or the partial pres- 
sure of atmospheric oxygen, is the factor ordinarily meant when 
oxygen requirement is mentioned. Many micro-organisms grow 
best in a medium freely exposed to the air. These are called 
aérobes. Some which will grow only when there is free access of 
oxygen are called obligate aérobes. There are numerous bacteria, 
including spirochetes, which grcw only in the absence of, or in 
extremely weak concentration of oxygen. These are called 
obligate anaérobes. Many of the bacteria grow well in various 
concentrations of oxygen or in its absence. These are spoken 
of as facultative anaérobes, or sometimes as facultative aérobes if 
they seem to prefer the anaérobic existence. Finally there are 
a few organisms, some bacteria and spirochetes, and perhaps some 
protozoa, which seem to require a fairly definite partial pressure 
of oxygen, but are not adapted to growth in a medium freely 
exposed to the atmosphere (B. bifidus, B. abortus, Spirocheta 
rossii, Plasmodium falciparum). In relation to oxygen require- 
ment, these are designated as microaérophilic organisms. 

Temperature.—Among the various micro-organisms are found 
types which are adapted for growth at different temperatures 
throughout a considerable range. There are some bacteria and 

‘perhaps some molds capable of growth at a temperature of —o.5° 
C.; in food substances such as milk, which are not frozen at this 
temperature. A certain yeast is said to multiply even at -6° C., 
in salted butter. Microbes which grow at very high temperatures, 
even up to 80° C., occur in the soil, in ensilage and sometimes 

‘in the intestine of animals. The great majority of micro-organ- 

isms grow only between o° and 40° C. It is possible to recognize ~ 

a minimum, a maximum and an intermediate optimum tem- 


174 GENERAL BIOLOGY OF MICRO-ORGANISMS 


’ 


perature for growth of each species. Ordinarily the optimum 
temperature is only a few degrees below the maximum at which 
growth will take place. The following table from Marshall's 
Microbiology illustrates the relation of these temperatures. 


& 


Temperatures 
{Species 
Minimum | Optimum Maximum 
Penicillium glaucum.......0 00.00 sivaSe 25°=27° 31°-36° 
Aspergillus niger......0.. cect eee cee 7°-10° 33°-37° 40°-43° 
Saccharomyces cerevisie I...........00. 1° 3° 28°-30° 40° 
Saccharomyces pasteurianus I............ 0.5° 25°-30° 34° 
Bacterium phosphoreum.............044. below 0° 16°-18° 28° 
Bacillus subtilis..... 0... eee 6° 30° 50° 
Bacterium anthracis..........0.0 0.000005 10° 30°-37° 43° 
Bacterium ludwigti..c... 6... c eee 50° 55°-57° 80° 


Heating above the maximum temperature for growth injures the 
microbe and exposure for a short time kills it. A temperature 
of 60° C. for 20 to 30 minutes destroys most vegetative forms of 
bacteria. Cooling, on the other hand, merely checks and inhibits 
growth. Freezing destroys some of the germs contained in a 
liquid but many of them remain alive. Still lower temperatures 
seem_to be entirely without further effect. Bacteria gradually 
die in frozen material. 

Germicides.— Unfavorable environmental factors, germicides. 
and antiseptics have been considered in an earlier Chapter 
(Chapter IT). 

Microbic Variation.—A microbic species is very stable in its 
characters when maintained under fairly constant conditions in 
its normal habitat. Change in environment brings about rather 
quickly change in some of the characters of a bacterial species. 
The alterations in virulence or ability to produce disease, which 
may be produced by methods of artificial culture, are perhaps best 
known. It would seem that, the descendants cf a single cell are 
not all identical, but they vary among themselves within fairly 


PHYSIOLOGY OF MICRO-ORGANISMS 175 


narrow limits in respect to a great many characters, fluctuating 
about a mean type which is that best adapted to the environment. 
With a change in surrounding conditions, this mean or normal 
type may no longer be kest adapted, but a variation slightly 
removed in respect to certain characters may flourish better and 
become the mean type about which the fluctuating variants group 
themselves. Thus the pure culture seems to respond to environ- 
mental change. Whether the fluctuating variations are due to 
small differences in the immediate surroundings of the individual 
microbes, or whether they arise as a result of a property of varia- 
bility inherent in protoplasm, may be disputed, but the latter 
view is more commonly held by biologists. 


THE Propucts oF MicroBic GROWTH 


The effects resulting from the growth of a micro-organism 
depend on the one hand upon the nature of the organism and on 
the other upon the environment, more especially the medium in 
which it grows and the conditions of temperature and oxygen 
supply. Apparently slight variations in the latter may influence 
the results to a marked degree. 

Physical Effects.—Heat is evolved by many actively growing 
bacterial cultures and is especially evident in the fermentation 
of such substances as ensilage and manure. Perhaps some of 
the heat may result directly from microbic activity, but the most 
of it appears to arise from secondary chemical reactions in which 
the microbic products sometimes play a part. Microbes which 
produce heat are designated as thermogenic. Light is also emitted 
by some microbic cultures. Here it seems certain that the light 
is produced by the oxidation of a bacterial product and not emitted 
directly by the micro-organisms. These phosphorescent or photo- 
genic organisms occur in salt water and on fish and they have 
rarely been found in other places. 

‘Chemical Effects.—These are the most important results of 
microbic growth. As we have just seen, the production of 
heat and light is probably due to a secondary reaction entered 


176 GENERAL BIOLOGY OF MICRO-ORGANISMS 


into by some of the chemical products of growth. Almost all 
the other important practical effects of the growth of micro- 
organisms are due to chemical changes produced by them. 
Primary products are those which are produced inside the cell 
by its living protoplasm. These include all the synthetic products 
such as the substance of the germ itself, the complex bodies 
which it forms from simpler substances, such as its enzymes 
and its toxins, and also the simpler chemical substances which 
result from internal cellular metabolism, the proper excretions 
of the cell. The secondary products are those which result from 
the action of a primary product, such as an enzyme, upon some 
material outside the cell. The distinction is clear enough in 
theory but practically it is often obscure. 

Enzymes.— Fermentation in its broad sense means the chemical 
changes brought about by living cells or their products. In its 
more restricted sense, it applies to the splitting of carbohydrates 
by the action of microbes, which is accompanied by_the evolution 
of gas. Organisms which cause active fermentation are spoken of 
as zymogenic. Dextrose, CeHi20s, is a readily fermentable 
carbohydrate and is decomposed in various ways by different 
microbes. In some instances a large proportion of it is converted 
into alcohol and carbon dioxide according to the following 
equation: 

CoH120o(fermented) = 2C2H,O + 2COs.. 


Other kinds of micro-organisms produce little alcohol or gas 
but abundant lactic acid. The reaction may be represented 
roughly by: this equation: 
Ce6H1206(fermented) = 2C3H,QOs. ai 
In other instances acetic acid may be produced: 
CeHi206(fermented) = 3C.H4Ox. 


These equations are only an approximate ‘indication of the re- 
actions which take place, as it is very doubtful that the whole 
molecule of dextrose is ever converted into a single simpler 


y 


PHYSIOLOGY OF MICRO-ORGANISMS 177 


compound by fermentation, but they will serve to indicate the 
nature of the reactions involved and to suggest the variety of 
products which may arise from the decomposition of complex 
organic substances. Some of these fermentative changes’ take 
place to a large extent inside the microbic cell. -Such is the 
case in the alcoholic fermentation produced by saccharomyces. 
The sugar-splitting or glycolytic ferments are found in the cultures 
of many bacteria and molds. Less common are the diastatic 
ferments capable of changing starch to dextrose, the inverting 
ferments which change saccharose and lactose into glucose and 
other hexoses, and the acetic ferments capable of causing the oxida- 
tion of alcohol to produce vinegar. 

The fermentation or decomposition of proteins usually gives 
rise to evil-smelling gases. This decomposition is called putrefac- 
tion, and the organisms which cause it are called. saprogenic. or 
putrefactive organisms. The nature of the products is much 
influenced by the amount of .oxygen available and the foulest 
gases are produced especially in the absence of oxygen. Proteo- 
lytic ferments of the same general nature as trypsin are produced 
by many microbes. A few form rennet-like enzymes. Proteo- 
lytic ferments which act in the presence of acid, like pepsin, are 
produced by some molds and by some bacterial species. 

The decomposition of the complex protein molecules gives rise 
to an enormous variety of intermediate products before the ulti- 
mate analysis into ammonia, carbon dioxide, water, sulphates and 
phosphates is accomplished. Many of these intermediate prod- 
ucts are very unstable and of unknown chemical’ composition. 
Some of them are highly poisonous. Brieger and his followers 
were able to separate a number of the complex substituted ammo- 
nia and ammonium compounds in a pure state and these par- 
ticular bodies are known as putrefactive alkaloids, or as flomains. 
A simple ptomain is trimethylamin, N(CHs)3; a more complex 
one cadaverin, HyN-CH2‘CH2:'CHz‘CH2‘CH2'NH2. Some of the 
ptomains are poisonous. These various decomposition products 


are for the most part secondary products resulting from the action 
12 


178 GENERAL BIOLOGY OF MICRO-ORGANISMS 


of enzymes upon the decomposing material. Many of them are 
so unstable that their presence in a decomposing substance is 
influenced by access of air, temperature and moisture, and they 
may quickly disappear or decompose. 

Micro-organisms also form fat-splitting or steatolytic enzymes, 
and enzymes capable of transforming urea into ammonium 
carbonate. 


NH2.CO.NH2+2H:20 (fermentation) = (NH4)2CO3. 


Various inorganic substances undergo chemical change under the 
influence of microbic activity and some of these changes appear to 
be due to enzymes. Specific examples will be considered in’ the 
section on the soil bacteria. — 

The toxins of bacteria are primary products built up by the cell. 
The true bacterial toxins are of unknown chemical composition, 
are labile like,enzymes and stimulate the production of antitoxins 
when they are injected into animals. They are the most poisonous 
substances at present known. Analogous substances have been. 
found in some plants, ricin in the castor bean and abrin in the 
jequirity bean, and the poisonous property of some kinds of snake 
venom is due to the presence of substances similar in nature to 
the bacterial toxins. These substances will be considered more 
fully in a later chapter devoted to the relation of parasitic microbes 
to their hosts. 


Murvuat RELATIONS OF A MICROBE AND ITS ENVIRONMENT 


Morphological Characters.—It is evident that the phenomena 
of growth taking place in a microbic pure culture depend not only 
upon the particular kind of microbe present but also in a very 
important way upon the chemical and physical structure of the 
medium, the access of air and the temperature. Variations in : 
these latter may even bring about considerable alteration in the 
form and structure of the individual cells. A common effect of 
high temperature is the shortening of individual bacilli and spirilla 
because of more rapid division and complete separation of the 


PHYSIOLOGY OF MICRO-ORGANISMS S Bee 


daughter cells. The presence of unfavorable influences, such as 
antiseptics or bacterial waste products in the medium, may cause 
marked irregularities in’ the shape and size of the cells, so-called 
involution forms. The ability to form endospores may be lost 
through growth at high temperature. The form which a micro- 
organisms presents in a given instance may not, therefore, be 
regarded as essentially typical without regard to the conditions 
under which it has been produced. ; 

The morphology of cell-groups is even more obviously depend- 
ent upon the conditions of the environment and the physiological 
properties of the micro-organism. A slow scanty growth on.a 
given medium does not necessarily mean that the organism 
essentially lacks vigor. It may mean that the medium is not well 
adapted to the requirements. Diffuse growth through a semi- 
solid medium may be merely an expression: of the motility of an 
organism. A great variety of different culture media have been 
employed to bring out more or less characteristic features in the 
gross appearance of cultures, and these appearances often depend 
upon the grouping of the cells or upon their fermentative activity 
or both. Although the characters of a cell-group of micro-organ- 
isms are really morphological characters of the same general na- 
ture as the morphological characters of higher plants and animals, 
to which so much significance is attached; in the case of micro- 
organisms in an artificial environment, such as a culture medium, 
the gross appearance or the cell-grouping is more properly regarded 
as a feature of physiological rather than morphological significance. 
Nutrient gelatin is a medium well adapted, in the case of those mi- 
crobes which will grow in it, for showing physiological differences 
in the appearance of cell-groups or colonies, and perhaps a greater 
variety of appearances may be obtained upon this medium than 
any other. Unfortunately its use entails certain difficulties, the 
most important of which is the necessity for experience and care 
in the interpretation of the appearances observed. Important 
features in the appearance of the colonies and other cell-groups 
are brought out by the use of various other media. 


180 GENERAL BIOLOGY OF MICRO-ORGANISMS 


Physiological Tests.—Specific tests for a simple physiological 
character require less skill and care in their observation, and are 
widely ysed. Cultivation in a fermentation tube of sugar broth 
as a test of ability to form gas from the sugar, titration of sugar- 
broth cultures to ascertain the ability to produce acid from various 
sugars, chemical test for the presente of indol and of ammonia ina 
culture in peptone solution, observation of the ability to hemolyze 
or discolor blood mixed with the medium, and the ability to fer- 
ment glycerin, these are some of the valuable simpler tests. 
Cultivation in milk is a somewhat more complex test, as a variety 
of fermentable substances is offered the microbe, increasing the 
difficulties of interpretation but also increasing the variety of phe- 
nomena which may occur. 

A convenient outline to use in making morphological and 
physiological observations upon bacteria and in recording the re- 
sults, has been prepared by'a committee of the Society of American 
Bacteriologists. Many features of this will be found of assistance 
in the study of new or unknown bacteria, especially saprophytic 
forms. A copy of the revised descriptive chart is inserted along 
with a copy of the earlier chart of 1907. 


DESCRIPTIVE CHART 
FOR USE IN BACTERIOLOGICAL INSTRUCTION 


Recommended by the Committee on the Chart for Identification of Bacterial Species at the 1917 meeting of the - J Coun ae 
I. J. KLIGLER Committee 
SOCIETY OF AMERICAN BACTERIOLOGISTS Ne ? Pisce bd 
K. N. ATKINS 
Name of organism........ f sp hbes ep ase Meda eyett os Sitio dL PHANG GS He ala ve den wR A Ae Rb al seme a8 Culttire: Noss icine cb bei dis ewees oe 
Source Dota c wig ipdniio oR adnan nines HES SESS RHE EGET Rete ES 


INVIGORATION OF CULTURE Date.......-.00005 
Series No.........-- 

Misa eee ar oo ovis No aie tas aA ee eats Hp aR ad Hila and SPE NST See eels al eecte tie 

Temperature.......... °C, Number of transfers........ 00 cee eee eee eee e eens 

Length of cach incubation... + ++e es crt ctr days. 

MORPHOLOGY 

ee So Oa, ee ee 
Nore—Underscore required terms. Sketches 


teMPay gaxeaye vers AGS ined Rigas Wee days. 
Form, spheres, short rods, long rods, filaments, com- 

mas, short spirals, long spirals, curved. 
Arrangement, single, pairs, chains, fours, cubical 


packets. 
Limits of Size.......... Size of Majority........- 
Ends, rounded, truncate, concave 
CAPSULES, Present ON... eee ee eee ee eee eee eet 


How stained. ....... cece eee eee eee nents 


Hea kg sea eave ABO arias goreheeileitas days. 
Form, elliptical, short rods, spindled, clavate, drum- 
Sticks. 
Limits of Size.......... Size of Majority........ 


ENDOSPORES, present, absent. 
Location of Endospores, central, polar. 
Form, spherical, elliptical, elongated. 
Limits of Size... .. ccc eee ee eee 
Size of Majority....... 5.0. e eee eee 
Wall, thick, thin. 
Sporangium wall, adherent, not adherent. 


i 


MOTILITY 
Tay brogh ys, cosa esd esee Om abate ay Ke enc eares 
FLAGELLA, No..........-- Attachment, polar, bipolar, 
peritrichiate. How stained.......-.--.++- 00ers 
IRREGULAR Forms. 
Present on.........- iti... days atric ev see. °C: 
Form spindled, cuneate, filamentous, branched, 
OE ota aha nn 2 iglesia Rese BRAY MOEA EL OES OTS 


STAINING REACTIONS. 
1:10 watery fuchsin, gentian violet, carbol fuchsin.) 
Loeffler’s alkaline methylene blue., | 
Special Stains. 
Gram esiss eg exsane cess Acid fast... 0.2 sc2e0 es es % 


Studied by 


Group No. 
GROUP NUMBER 
As each of the determinations listed below is made. 
check the proper figure. When complete place the entire 
group number in the spacc above. 
100.0 Endospores produced 
200.0 Endospores not produced 
10.0 Aerobic (Strict) 
20.0 Facultative anaerobic 
30.0 Anaerobic (Strict) 
I.0 Gelatin liquefied 
2.0 Gelatin not liquefied 
o.I Acid and gas from dextrose 
0.2 Acid without gas from dextrose 
0.3 No acid from dextrose 
0.4 No growth with dextrose 
0.0r Acid and gas from lactose 
0.02 Acid without gas from lactose 
0.03 No acid from lactose 
0.04 No growth with lactose 
0.001 Acid and gas from saccharose 
0.002 Acid without gas from saccharose 
0.003 No acid from saccharose 
0.004 No growth with saccharose 
0.0001 Nitrates reduced with evolution of gas 
0.0002 Nitrates reduced without gas 
0.0003 Nitrates not reduced 
0.00001 Fluorescent 
0.00002 Violet chromogens 
0.00003 lue chromogens 
0.00004 Green chromogens 
0.00005 Yellow chromogens 
0.00006 Orange chromogens 
0.00007 Red chromogens 
0.00008 Brown chromogens 
0.00009 Pink chromogens 
0.00000 Non-chromogenic 
0.000001 Diastatic action on starch, strong 
0.000002 Diastatic action on starch, feeble 
0.000003 Diastatic action on starch, absent 
0 .0000001 Acid and gas from glycerin 
0 .0000002 Acid without gas from glycerin 
0 .0000003 No acid from glycerin 
0 .0000004 No growth with glycerin 


BRIEF CHARACTERIZATION 


The genus according to the system of Migula is given 
its proper symbol which precedes the number thus: 


BACILLUS COLI (Esch.) Mig. 


becomes B. 222.111202 


i; 


Diameter over Iu 


Diameter 0.5-Iu 


Diameter under 0.54 


Length over two diams. 


Chains (4 or more cells) 


Filaments 


Capsules 
Motile 


Gram’s Stain 


STIGO AALLVLEOTA 
| 


Central 


Polar 


Diameter > Diam. of rod 
Round 
Oval to cylindrical 


| sHaodsSOaNa 
| 


Abundant 
Absent 
Shining 
Wrinkled 


Chromogenic 


Punctiform 


Round (over I mm.) 
Rhizoid 
Filamentous 

Curled 


Punctiform 


SaUNLVad TVaAALTINAO 


ee Ores Ne eee 
Round (over 1 mm.) 
Pica tahoe nas alam 


Irregular 


"Filamentous 
Acid curd 


Rennet curd 


s Casein n peptonized 
= | 


Unchanged 


| Fluorescent 


Plant pathogen 
icin creed aia Deon eee 


| 
| Saprophyte 


CULTURAL CHARACTERISTICS 


Changes, ..-+- Changes, ........4. days \KETCHES 
Growth, scanty, moderate, abundant, none. 
Agar Form of growth, filiform, echinulate, beaded, spread- : 
Stroke ing, arborescent, rhizoid. 
Elevation of growth, flat, effuse, raised, convex. | 
Lustre, glistening, dull. } 
Incubation Topography, smooth, contoured, rugose. \ 
Temperature Optical Characters, opaque, translucent, opalescent, ! 
tridescent. 
Chromogenesis.......... Photogenic. Fluorescent. | 
cpnswsne SCS Odor, absent, decided, resembling.................. 
Consistency, butyrous, viscid, membranous, britile. 
Medium, grayed, browned, reddened, blued, greened. 
Growth, uniform, best at top, best at butiom. ; 
Gelatin Line of puncture, filiform, beaded, papillate, villous, | 
Stab arborescent. 
Liquefaction, none, crateriform, napiform, infundi- 
buliform, saccate, stratiform; begins in......... \ 
Temperature complete in............ d. | 
Depth of liquefaction in tube of 10 mm. diameter | 
Sdethe Sie °C. evenly inoculated at 20° C. for 30 days.....mm. i | 
Medium, fluorescent, browned. 
| 
1 $< $$$ 
| | 
Medium | 
(solid) | | 
Temperature | ! 
Hee ene °C. | | 
Lael tde hours Changes, ......days Changes, ......days oe feeaens hours Changes, ......days Changes, ......days 
Nutrient Surface growth, ring, pellicle, flocculent | eee 
Broth membranous, none. ! Medium 
Clouding, slight, moderate, strong tran- ! (liquid) 
sient, persistent, none, fluid turbid. 
Temperature | Odor, absent, decided, resembling........ 
Sediment, compact, flocculent, granular, | Temperature 
flaky, viscid on agitation, abundant, 1} ° 
eenees °c. scant, none. ; l ee a 
SKETCHES 
Surface Colony Deep Colony 
Growth, slow, rapid. 
Agar Form, punctiform, circular, irregular, mycelioid, 
Colonies filamentous, rhizoid. 
Surface, smooth, rough, concentrically ringed, radiate. 
: Elevation, fiat, effuse, raised, convex, pulvinate, 
Temperature umbonate. 
Edge, entire, undulate, lobate, erose, filamentous, 
curled. I 
ein eae °c. Internal structure, amorphous, finely-, coarsely-granu- | | 
lar, filamentous, curled, concentric. | ..days | .. days ..days . days 
Growth, slow, rapid. 
Gelatin Form, punctiform, circular, irregular, mycelioid, 
Colonies filamentous. 
Elevation, flat, raised, convex, pulvinate, crateriform 
(liquefying). 
Temperature | Edge, entire, undulate, lobate, erose, filamentous, 
floccose, curled. os 
Liquefaction, cup, saucer, spreading. 
eer °C. Internal structure, amorphous, finely-, coarsely- 
granular, filamentous, curled, concentric. .. days .. days .. days . days 


— 


Source... 


Date of Revivifaction. 


Pe cecccccc cece cers ceceeeeeesereccsesees 


Date of Isolation. ...........ccceecesesnceeeeeeereees 


.. Determined: When ?............ 


NAME: sicisis oississisisicciecesieis a2 dine 


By whom? ...... 


Group No.... . 
Culture No 


DETAILED FEATURES 


GELATIN Stas. 


NoTE—Underscore required"terms. Observe notes. 
VEGETATIVE CELLS, Medium used.........ees seers 
TEMP ose ew sgccces $ AMC oi si05 ees HOES - 
Form,'spheres,*short rods, long rods, filaments, com- 
mas, short spirals, long spirals, spindled, cuneate, 
clavate, curved. 
Arrangement, single, pairs, chains, fours, cubical 
packets. 
Limits of Size..... 
Size of Majority 
Ends, rounded, truncate, concave. 
Orientation (grouping) 
Chains (No. of elements)........ 
Orientation of Chains, parallel, 
irregular. 
Postfission movements, loop forming, folding, snap- 
ping, slipping. 
SPORANGIA, present, absent. 


Agar 
Hanging-Block 


Medium used.......... 


PONIPs os So sass 5s sek ABCs aa eee awsie days. 
Form, elleptical, short rods, spindled, clavate, drum- 
Sticks. 
Limits of Size.......... Size of Majority... 
Orientation (grouping).... Z 
Agar Chains (No. of elements)........ 


Orientation of Chains, parallel, 
irregular. 
ENDOSPORES, present, absent. 
Location of Endospores, central, polar. 
Form, spherical, elliptical, elongated. 
Limits of Size... 
Size of Majority 
Wall, thick, thin. 
Sporangium wall, adherent, not adherent. 


Hanging-Block 


Committee on Revision of Chart Identification of Bacterial Species. 


Germination, equatorial, oblique, polar, bipolar, 
by stretching, by absorption of spore wall. 
——~—— CAPSULES, present On.......... eee 
ZOoGLOEA, Pseudozoogloea. . 

9 ¢ FLAGELLA, No......... Attachment, polar, bipolar, 
Z me SI lophotrichiate, peritrichiate. _How Stained....... 
2288 InvoLurion Forms, on........ in...days at..... °C. 
< S @&2 STAINING REACTIONS. . . 
LO Ama, 1:10 watery fuchsin, gentian violet, carbol fuchsin, 
aad Loeffler’s alkaline methylene blue. 
+ K-.: Special Stains. 
Hh Ba « Gram chs 6 bk eases Glycogen. .........45 
b Fat..... -Acid fast..........04. 
a NEGISSET Sc) isieiesis = gree tens AS 
cs) Metachromatic granules, sporogenous granules. 
& NUTRIENT BrotH. 
g Surface growth, ring, pellicle, flocculent, membran- 
2 ous, none. : 
A Clouding, slight, moderate, strong, transtent, per- 


sistent, none, fluid, turbid. 
Odor, absent, decided, resembling......+++.-++200% 
Sediment, compact, flocculent, granular, flaky, viscid 
on agitation, abundant, scant, none. 
AGAR STROKE. 
Growth, invisible, scanty, moderate, abundant, none. 
Form of growth filiform, echinulate, beaded, spread- 
ing, plumose, arborescent, rhizoid. 
Elevation of growth, flat, effuse, raised, convex. 
Lustre, glistening, dull, cretaceous. 
Topography, smooth, contoured, rugose, verrucose. 
Optical Characters, opaque, translucent, opalescent, 
tridescent. 
Chromogenesis........- 
Odor, absent, decided, resembling. ......0cesceeeee 
Consistency, slimy, butyrous, viscid, membranous, 


Photogenic. Fluorescent. 


& 
fo 
< 
i = coriaceous, brittle. 
9 Q Medium, grayed, browned, reddened, blued, greened. 
S - AGAR COLONIES. 
Ha ad Growth, slow, rapid, temperature..........045 se 
A eo Form, punctiform, circular, irregular, ameboid, 
1 Ss mycelioid, filamentous, rhizoid. 
& Q Surface, smooth, rough, concentrically ringed, 
n radiate, striate. 
Q Elevation, flat, effuse, raised, convex, pulvinate, 
A umbonate. 

Edge, entire, undulate, lobate, erose, lacerate, 


jfimbriate, floccose, curled. 
Internal structure, amorphous, finely-, coarsely- 
granular, grumose, filamentous, floccose, curled. 
GELATIN COLONIES. 
Growth, slow, rapid. 
Form, punctiform, circular, irregular, 
mycelioid, filamentous, rhizoid. 
Elevation, flat, effuse, raised, convex, pulvinate, 
crateriform (liquefying). 
Edge, entire, undulate, lobate, erose, 
_Simbriate, filamentous, floccose, curled, 
Liquefaction, cup, saucer, spreading, 


ameboid, 


SOCIETY OF AMERICAN BACTERIOLOGISTS 


Endorsed by the Society for General Use at the Annual Meeting 


lacerate, 


Growth, uniform, best at top, best at bottom. | 

Line of puncture, filiform, beaded, papillate, villous, 
plumose, arborescent. L : 

Liquefaction, none, crateriform, napiform, wien 


buliform, saccate, startiform; begins in........- 5 
complete in: e+ ..seqs ees d. i 
Depth of liquefaction in tube of 10 mm. diameter, 
evenly inoculated at 20°C. for 30 days..... mm. 
POTATO. 
Growth, scanty, moderate, abundant, transient, 
persistent. 


Form of growth, filiform, echinulate, beaded, 
spreading, plumose, arborescent, rhizoid. 

Elevation of growth, flat, effuse, raised, convex. 

Lustre, glistening, dull, cretaceous. 

Topography, smooth, contoured, rugose, verrucose. 

Chromogenesis........+-+e20e05 Pigment in water 
insoluble, soluble; other solvents........+++ee05 

Odor, absent, decided, resembling. .....00.0eeeeeee 

Consistency, slimy, butyrous, viscid, membranous, 
coriaceous, brittle. 

Medium grayed, browned, reddened, blued, greened. 


Potato STARCH JELLY. 
Growth, scanty, copious, absent. 
Diastasic action, absent, feeble, profound. 
Medium stained 
Dextrin, present, absent. 


TEMPERATURE RELATIONS. 


Optimum SepEMa tune for growth 20°C.: or 37°C.: 
[) ere aed . , 

Maximum temperature for growth..........-- °C. 

Minimum temperature for growth...........- en 


Coun’s SOLUTION. 


Growth, copious, scanty, absent. 
Medium fluorescent, nonfluorescent. 


UScHINSKY’s SOLUTION. 


Growth, copious, scanty, absent. 
Fluid, viscid, not viscid. 


LoEFFLER’s BLoop SERUM. 


Stroke invisible, scanty, moderate, abundant. 
Form of growth, filiform, echinulate, 
spreading, plumose, arborescent, rhizoid. 
Elevation of growth, flat, effuse, raised, convex. 
Lustre, glistening, dull, cretaceous. 
Topography, smooth, contoured, rugose, verrucose. 
Chromogenesis 
Medium grayed, browned, reddened, blued, greened. 
Liquefaction begins in .....d., complete in..... d. 


MILK. 


Clearing without coagulation. 

Coagulation prompt, delayed, absent. 

Extrusion of whey begins in............ days. 
Peptonization begins on....d., complete on..... d. 
Coagulum, slowly peptonized, rapidly peptonized. 
Reaction, Id.... 2d.... . Iod.... 20 
Consistency, slimy, viscid, unchanged. 
Medium browned, reddened, blued, greened. 


beaded, 


Litmus Mik. 


Acid, alkaline, acid then alkakine, no change. 
Prompt reduction, no reduction, partial slow re- 
duction. 


InDoL Propuction, feeble, moderate, strong, absent. 


HypRoGEN SULPHDIE, feeble, moderate, strong, ab- 
sent. 


AMMONGIA PRODUCTION, feeble, moderate, strong, ab- 
sent, masked by acids. 
NITRATE IN NITRATE BrotH. Reduced, not reduced. 
Presence of nitrites........ Ammonia 
Presence of nitrates.........free nitrogen. . 


NITROGEN. Obtained from peptone, 


te asparagin, 
glycocoll, urea, ammonia salts, nitrogen. 


SILICATE JELLY (Fermi’s Solution). 
Growth, copious, scanty, absent. 


Medium stained.............005 
Best media for long-continued growth............ 


Endospores produced 100, 
Endospores not produced 200. 


Aerobic (Strict) 10. Facultative anaerobic 20. 
Anaerobic (Strict) 30. 


Gelatin liquefied 1. 
Gelatin not liquefied 2. 


Acid and gas .1. Acid without gas .2. 


No acid .3. No growth .4 with dextrose. 
Acid and gas .or. Acid without gas .02. 
No acid .03. No growth .04 with lactose. 


Acid and gas .oor. Acid without gas .002. 
No acid .003. No growth .004 with saccharose. 


Nitrates reduced with gas .ooo1. 

Nitrates reduced without gas .0002. 

Nitrates not reduced .0003. 

Fluorescent .o0001. Violet, .00002. 
Green .00004. Yellow .oo005. Orange .00006. 
.00007. Brown .00008. Pink .oo009 =Non-chromo- 
genic .00000. 

Diastasic acticn on potato starch, strong .000001 

Feeble .o00002. Absent .000003. 


Blue .00003 


Red 


Acid and gas .oooo001. Acid without gas .0000002 
Noacid .0000003. No growth .0000004 with glycerin. 


1. Fermentation-tubes con- 
taining peptone-water or 
sugar-free bouillon. 

Abicaiensie’s °C. 


Dextrose 
Lactose 
Saccharose 
sGlycerin 


Gas production, in per cent 


H 
(co:) 
Growth in closed arm 
Amount cf acid produced d. 


Amount of acid produced d. 
Amount of acid produced d. 


PATHOGENIC TO ANIMALS. 
Insects, crustaceans, fishes, reptiles, birds, mice, 


rats, guinea pigs, rabbits, dogs, cats, sheep, 
goats, catile, horses, monkeys, MAN....+.++++0005 
Serum reactions, agglutinins, bactericidins, opsonins, 
precipitins, complement deviation.........+++++5 


Products of growth, soluble toxins, endotoxins. 


PATHOGENIC TO PLANTS. 
Plants affected. ..... cece eee erence ences : 


Loss oF VIRULENCE ON CULTURE MEDIA, prompt, 
gradual, not observed in.........0000 months. 

Acips PRopUCED 
ALKALIES PRODUCED. 
ALCOHOLS PRODUCED 


FERMENTS: peptase, iryptase, chymase, diastase, 
invertase, pectase, cytase, tyrosinase, oxidase, 
peroxidase, reductase. catalase, lipase, glucase, 
galactase, CtC... cee eee eee eee eee eee e nee 

Crytals formed:.. 4... 0. cece eee cere eee e en ne nee 

Toleration of Acids: Great, medium, slight. 

Acids tested. .cceeccscrecenee 


Toleration of Alkalies: Great, medium, slight. 

Alkalies Tested.........cec000 

Optimum reaction for growth in bouillon, stated 
in terms of Fuller’s scale 

Killed readily by drying: resistant to drying. 

Per cent killed by freezing (salt and crushed ice 
or liquid air) 

Sunlight: Exposure on ice in thinly sown agar 
plates: one-half plate covered (time 15 minutes), 
Sensitive, not sensitive. 

Per cent killed : : 

Vitality on culture media: brief, moderate, long. 

Thermal death-point (10 minutes exposure in nu- 
trient broth when this is adapted to growth of 
organism) °C, 


BRIEF CHARACTERIZATION 


ADOTOHAIOW 


SGYNLVad TWAALTAO 


SHUALVa TVOINGHOOIA ~— | 


NOILNGIULSIA 


Diameter over 1% 


Chains, filaments 


Endospores 


Capsules 


Zoogloea, Pseudozoogloea 
Motile 


Involution forms 


Gram’s Stain 


Cloudy, turbid 
4 Ring a 
§ | Pellicle 
Sediment 
Shining 
> | Dull me 
| Wrinkled 
Chromogenic oa 
Round baal 
© | Proteus-like 
7 Rhizoid 
= Filamentous 
Curled 
#2 Surface-growth 
o | Needle-growth 
oe Moderate, absent at 
9 | Abundant a 
= Discolored ‘aed 
Starch destroyed a 


Grows at 37°C. 

Grows in Cohn’s Sol. 
Grows in Uschinsky’s Sol. 
Gelatin 

Blood-serum 


Casein 


Agar, mannan 
Acid curd 
Rennet curd 


AW [Boroeyenbry 


Casein peptonized 
Indol. 
Hydrogen sulphide 


Ammonia 
Nitrates reduced 


Fluorescent 


Luminous 

Animal pathogen, epizoon 
Plant pathogen, epiphyte 
Soil 

Milk 

Fresh water 


Salt water 


Sewage 
Iron bacterium 
Sulphur bacterium 


PHYSIOLOGY 


FERMENTATION MILK 
Temperature............ °c. 
Temperature............ °C. 
a j REACTION COAGULATION PEPTONIZATION 
Fermentation-tubes a 8 x | ! 
containing........... g a a re | ih 
a cs ee ee a g = a | i PAM ay eas cnevad cvedwetarrsaeaas DAY ie wieiron ewan oo shins ace 3X 0s 
aSloleinit hi sins e's Sus Bier age tes iM i o ba r DAC AYS see at Vda winders 6 ecesecare Bday sacs oy ees UF os sac evens 8 ao, 
Wie ad Bove Ghee ead Ses and: A < 4 3 | A SAY Ss -siece sigs she ere covchentte fe a Wekearas MAYS tisSieis5s 575-4. danannoa awa 
VL, TP AAY Ss vewaw senso east os he Cais FT GAYS rare GoeE Bian cideunncs aseethiase 
{ UOLCAY S: samadeta stow wis cetengees TO.days. 1.8 POM, oxas cass 
Growth in closed arm ! 
| 
LITMUS MILK 
oO; 
ERE 2 Temperature............ Cc. 
7 REACTION COAGULATION PEPTONIZATION REDUCTION 
Coz Epes 
1 day [SB Ale is wwe his usenet CL ee ee eee Oey stds ies ees 
2 days BAA Srocice saa. shoe Wes POYSsc.eeased caus 2 days.. 
ACH 1, cathe aks days 4 days AAV Sess sesie pielartiae's ro er ee er 4 days 
7 days BAAS tara wcaie e:dsters- acs a F OVS hobs hos eden e D GEV S fois erase Sb whee 
10 days , 10 (cea TO GAYS... Vseras ote ola « 10 dBYS.(.. 2. 0c ce Ceres 
Acid tint nis seins days 
NITRATE REDUCTION 
WACO iy na saab oad cioks paw a ee Temperature............ °c. 
MOTE Tice css sees days Nitrite: 1day........... PR GAYS: teaavices on PAdays ee ecient SP AY SK ts teacgiae odes PTO MAYS seicsacace ian, 
Gas: Dia ye ris occ seg aracais FRO BYS ioe boston enn $A GAYS«. 6 veiecheen 37 days...... wie LO GAYS cess sie aden 
CHROMOGENESIS DIASTATIC ACTION 


Nutrient broth 


Breadth of clear zone on starch agar plates 


i a we ee BBitcibiceeeipeldisceee saa days 
Nutrient gelatin in y 
AT arbi vets hdisan cay tc. Ss NS days 
Nutrient agar 
TOS ccaiene g's ssekiaiarede meee ae days 


Potato 


TEMPERATURE RELATIONS 
Optimum temperature for growth....... ccc cee cece e cee cee eee eee 


Maximum temperature for growth 


Minimum temperature for growth 


DESCRIPTIVE CHART—SOCIETY OF AMERICAN BACTERIOLOGISTS 
Prepared by F. D. Chester, F. P. Gorham, Erwin F. Smith, Committee on Methods of Identification of Bacterial Species. 


Endorsed by the Society for general use at the Annual Meeting, Dec. 31, 1907. 


GLOSSARY OF TERMS 


AGAR HANGING-BLOCK, a small block of nutrient agar cut from 
a poured plate, and placed on a cover-glass, the surface next 
the glass having been first touched with a loop from a young 
fluid culture or with a dilution from the same. It is examined 
upside down, the same as a hanging drop. 

AMEBOID, assuming various shapes like an ameba. 

AMORPHOUS, without visible differentiation in structure. 

ARBORESCENT, a branched, tree-like growth. 

BEADED, in stab or stroke, disjointed or semi-confluent colonies 
along the line of inoculation. 

BRIEF, a few days, a week. 

BRITTLE, growth dry, friable under the platinum needle. 

BULLATE, growth rising in convex prominences, like a blistered 
surface. 

BUTRYOUS, growth of a butter-like consistency. 

CHAINS, 

Short chains, composed of 2 to 8 elements. 
Long chains, composed of more than 8 elements. 

CILIATE, having fine, hair-like extensions like cilia. 

CLOUDY, said of fluid cultures which do not contain pseudozoogloeae. 

COAGULATION, the separation of casein from whey in milk. This 


may take place quickly or slowly, and as the result either of the 
formation of an acid or of a lab ferment. 

CONTOURED, an irregular, smoothly undulating surface, like that 
of a relief map. 

CONVEX, surface the segment of a circle, but flattened. 

COPROPHYL, dung bacteria. 

seagate eu growth tough, leathery, not yielding to the platinum 
needle. 

CRATERIFORM, round, depressed, due to the liquefaction of the 
medium. 

CRETACEOUS, growth opaque and white, chalky. 

CURLED, composed of parallel chains in wavy strands, as in anthrax 
colonies. 

DIASTASIC ACTION, Same as DIASTATIC, conversion of starch 
into water-soluble substances by diastase. 

ECHINULATE, in agar stroke a growth along line of inoculation, 
with toothed or pointed margins; in stab-cultures growth beset 
with pointed outgrowths. 

EFFUSE, growth thin, veily, unusually spreading. 

ENTIRE, smooth, having a margin destitute of teeth or notches. 

EROSE, border irregularly toothed. 

FILAMENTOUS, growth composed of long, irregularly placed or 
interwoven filaments. 

FILIFORM, in stroke or stab-cultures a uniform growth along line 
of inoculation. 

FIMBRIATE, border fringed with slender processes, larger than 
filaments. 

FLOCCOSE, growth composed of short curved chains, variously 
oriented. 


FLOCCULENT, said of fluids which contain pseudozoogloeae, i.e., 
small adherent masses of bacteria of various shapes and floating 
in the culture fluid. 

FLUORESCENT, having one color by transmitted light and another 
by reflected light. 

GRAM’S STAIN, a method of differential bleaching after gentian 
violet, methyl violet, etc. The -++ mark is to be given only 
when the bacteria are deep blue or remain blue after counter- 
Staining with Bismark brown. 

GRUMOSE, clotted. 

INFUNDIBULIFORM, form of a funnel or inverted cone. 

IRIDESCENT, like mother-of-pearl. The effect of very thin films. 

ERE RATE, having the margin cut into irregular segments as if 

orn. 


LOBATE, border deeply undulate, producing lobes (see undulate). 


LONG, many weeks, or months. 

MAXIMUM TEMPERATURE, temperature above which growth 
does not take place. 

MEDIUM, several weeks. 

MEMBRANOUS, growth thin, coherent, like a membrane. 

MINIMUM TEMPERATURE, temperature below which growth 
does not take place. 

MYCELIOID, colonies having the radiately filamentous appear- 
ance of mold colonies. 

NAPIFORM, liquefaction with the form of a turnip. 

NITROGEN REQUIREMENTS, the necessary nitrogenous food. 
This is determined by adding to nitrogen-free media the nitrogen 
compound to be tested. 

OPALESCENT, resembling the color of an opal. 

OPTIMUM TEMPERATURE, temperature at which growth is 
most rapid. 

PELLICLE, in fluid bacterial growth either forming a continuous 
or an interrupted sheet over the fluid. 

PEPTONIZED, said of curds dissolved by trypsin. 

PERSISTENT, many weeks, or months. 

PLUMOSE, a fleecy or feathery growth. 

PSEUDOZOOGLOEAE, clumps of bacteria, not dissolving readily 
in water, arising from imperfect separation, or more or less 
fusion of the components, but not having the degree of com- 
pactness and gelatinization seen in zopgloeae. 

PULVINATE, in the form of a cushion, decidedly convex. 

PUNCTIFORM, very minute colonies, at the limit of natural vision. 

RAISED, growth thick, with abrupt or terraced edges. 

RHIZOID, growth of an irregular branched or root-like character, 
as in B. mycoides. 

RING, Same as RIM, growth at the upper margin of a liquid culture, 
adhering more or less closely to the glass. 

REPAND, wrinkled. 

RAPID, developing in 24 to 48 hours. 

SACCATE, liquefaction the shape of an elongated sack, tubular, 
cylindrical. 

SCUM, floating islands of bacteria, an interrupted pellicle or bacterial 
membrane. 

SLOW, requiring 5 or 6 days or more for development. 

SHORT, applied to time, a few days, a week. 

SPORANGIA, cells containing endospores, 

SPREADING, growth extending much beyond the line of inoculation, 
t.e., several millimeters or more. 

STARTIFORM, liquefying to the walls of the tube at the top and 
then proceeding downward horizontally. 

THERMAL DEATH-POINT, the degree of heat required to kill 
young fluid cultures of an organism exposed for 10 minutes (in 
thin-walled test-tubes of a diameter not exceeding 20 mm.) 
in the thermal water-bath. The water must be kept agitated 
so that the temperature shall be uniform during the exposure. 

TRANSIENT, a few days. 

TURBID, cloudy with flocculent particles; cloudy plus flocculence. 

UMBONATE, having a button-like, raised center. 

UNDULATE, border wavy, with shallow sinuses. 

VERRUCOSE, growth wart-like, with wart-like prominences. 

VERMIFORM-CONTOURED, growth like a mass of worms, or in- 
testinal coils. 

VILLOUS, growth beset with hair-like extensions. 

VISCID, growth follows the needle when touched and withdrawn, 
sediment on shaking rises as a coherent swirl. 

ZOOGLOEAE, firm gelatinous masses of bacteria, one of the most 
typical examples of which is the Streptococcus mesenterioides 
of sugar vats (Leuconostoc mesenterioides) the bacterial chains 
being surrounded by an enormously thickened firm covering 
inside of which there may be one or many groups of the bacteria. 


NOTES 


(1) For decimal system of group numbers see Table 1. This will 
be found useful as a quick method of showing close relationships inside 
the genus, but is not a sufficient characterization of any organism. 

(2) The morphological characters shall be determined and described 
from growths obtained upon at least one solid medium (nutrient agar) 
and in at least one liquid medium (nutrient broth). _Growths at 37°C. 
shall be in general not older than 24 to 48 hours, and growths at 20°C. 
not older than 48 to 72 hours. To secure uniformity in cultures, in 
all cases preliminary cultivation shall be practised as described in the 
the revised Report of the Committee on Standard Methods of the 
Laboratory Section of the American Public Health Association, 1905. 

(3) The observation of cultural and bio-chemical features shall 
cover a period of at least 15 days and frequently longer, and shall be 
made according to the revised Standard Methods above referred to. 
All media shall be made according to the same Standard Methods. 

(4) Gelatin stab-cultures shall be held for 6 weeks to determine 
liquefaction. | . 

_(5) Ammonia and indol tests shall be made at end of 1oth day, 
nitrite tests at end of 5th day. 


(6) Titrate with 35 NaOH, using phenolphthalein as an indicator: 


make tirations at same times from blank. The difference gives the 
amount of acid produced. 

The titation should be done after boiling to drive off any CO2 
present in the culture. A 

(7) Generic nomenclature shall begin wtih the year 1872. (Cohn’s 
first important paper. 

Species nomenclature shall begin with the year 1880. (Koch’s dis- 
covery of the poured plate method for the separation of organisms.) 

(8) Chromogensis shall be recorded in standard color terms. 


TABLE I. 


A NUMERICAL SYSTEM OF RECORDING THE SALIENT 
CHARACTERS OF AN ORGANISM. (GROUP NUMBER) 


I00.0 Endospores produced 
200.0 Endospores not produced 
10.0 Aérobic (Strict) 
20.0 Facultative anaérobic 
30.0 Anaérobic (Strict) 
° Gelatin liquefied 
.0 Gelatin not liquefied 
I Acid and gas from dextrose 
2 Acid without gas from dextrose 
3 No acid from dextrose 
4 No growth with dextrose 


or Acid and gas from lactose 
02 Acid without gas from lactose 
03 No acid from lactose 
04 No growth with lactose 
oor Acid and gas from saccharose 
002 Acid without gas from saccharose 
.003 No acid from saccharose 
.004 No growth with saccharose _ 
Nitrate reduced with evolution of gas 
Nitrates not reduced . 
Nitrates reduced without gas formation 
Fluorescent 
Violet chromogens 
Blue chromogens 
Green chromogens 
Yellow chromogens 
Orange chromogens 
Red chromogens 
Brown chromogens 
Pink chromogens 
Non-chromogenic 
Diastasic action on potato starch, strong 
Diastasic action on potato starch, feeble 
Diastasic action on potato starch, absent 
Acid and gas from glycerine | 
Acid without gas from glycerine 
.0000003. No acid from glycerine 
0000004 No growth with glycerine 
The genus according to the system of Migula is given its proper 
symbol which precedes the number thus: (7) 


.OO0O0O00T 
000002 

-000003 

.000000L 
0000002 


SCODSDHDDDODDCDODSCODGCOSOSOSCOODOCOOOOOCOOONH 
° 
° 
° 
° 
4 


BacILus cou! (Esch.) Mig. becomes B. 222.111102 
BACILLUS ALCALIGENES Petr. becomes B. 212 .333102 
PSEUDOMONAS CAMPESTRIS (Pam.) Sm. becomes Ps.  211.333151 


BACTERIUM SUICIDA Mig. becomes Bact. 222.232203 


Source.......-e cece cece eee eereee iad MANERA Re LEER 


NOTE.—Underscore required terms. 


DETAILED FEATURES 
Observe notes 


and glossary of terms on opposite side of card. 


I. 


eyo 


1%. 
. Agar Stroke. 


MORPHOLOGY (?) 


tem 

Form, round, short 
long chains, filaments, commas, short spirals, long 
spirals, clostridium, cuneate, clavate, curved. 


Limits of Size........- 

Size of Majority.........- 

Ends, rounded, truncate, concave. 

Orientation (grouping)....... 

Chains (No. of elements).... 

Short chains, long chains 

Orientation of Chains, parallel 
irregular. 


Agar 
anging-block 


Size of Majority........ 

{ Orientation (grouping)....... 
Chains (No. of elements).... 
Orientation of Chains, parallel, 
irregular. 

Location of Endospores, central, polar. 


Agar 
Hanging-block 


. Endospores. 


Form, round, elliptical, elongated. 

Limits of Size.... 

Size of Majority. 

Wall, thick, thin. 

Sporangium wall, adherent, not adherent. 

Germination, equatorial, oblique, polor, bipolar, by 
stretching. 


. Flagella No....... Attachment polar, bipolar, per- 


itrichiate. How Stained............ 


Capsules, present on......... 

. Zoogloea, Pseudozoogloea. 

. Involution Forms, on...... te ears days at..... °C. 

. Staining Reactions. 

1:10 watery fuchsin, gentian violet, carbol fuchsin, 
Léffler’s alkaline methylene blue. 

Special Stains 


Glycogen..........6. 
Acid fast... cc00s csucsie 


CULTURAL FEATURES (8) 


Growth, invisible, scanty, moderate, abundant. 

Form of growth, filiform, echinulate, beaded, spreading 
plumose, arborescent, rhizoid. 

Elevation of growth, flat, effuse, raised, convex. 

Luster, glistening, dull, cretaceous. 

Topography, smooth, contoured, rugose, verrucose. 

Optical Characters, opaque, translucent, opalescent, 
aridescent. 

Chromogenesis (8)........-. 

Odor, absent, decided, resembling........ 

Consistency, slimy, butyrous. viscid, membranous, 
coriaceous, brittle. 

Medium grayed, browned, reddened, blued, greened. 


. Potato. 


Growth, scanty, moderate, abundant, transient, per- 
sistent. 

Form of growth, filiform, echinulate, beaded, spreading, 
plumose, arborescent, rhizoid. 

Elevation of growth, flat, effuse, raised convex. 

Luster, glistening, dull, cretaceous. 

Topography, smooth, contoured, rugose, verrucose. 

Chromogenesis (8) Pigment in water 
insoluble, soluble; other solvents...........00000+ 

Odor, absent, decided, resembling..........00 ee ences 

Consistency, slimy, butyrous, viscid, membranous, 
coriaceous, brittle. 

Medium grayed, browned, reddened, blued, greened. 


. LéMler’s Blood Serum. 


Stroke, invisible, scanty, moderate, abundant. Form 
of growth, filiform, echinulate, beaded, spreading, 
plumose, arborescent, rhizoid. 

Elevation of growth flat, effuse, raised, convex. 


. Agar Stab. 


Growth uniform, best at top, best at bottom; surface 
growth scanty, abundant; restricted, wide-spread. 

Line of puncture, filiform, beaded, papillate, villous, 
plumose, arborescent; liquefaction. 


10. 


Il. 


12. 


13. 


14. 


15. 
16. 
17. 
18. 


. Gelatin Stab. 


Growth uniform, best at top, best at bottom. ¢ 
Line of puncture, filiform, beaded, papillate, villous, 
plumose, arborescent. 


Liquefaction, crateriform, napiform, infundibult- 
form, saccate, startiform; begins in.......-++0--- d, 
complete in.......++--- d. 


. Nutrient Broth. 


Surface growth, ring, pellicle, flocculent, membranous, 


none. 

Clouding slight, moderate, strong; transient, persistent; 
none; fluid turbid. 

Odor, absent, decided, resembling.........-++ 

Sediment, compact, flocculent, granular, 
on agitation, abundant, scant. 


. Milk. 


Clearing without coagulation. 

Coagulation prompt, delayed, absent. 

Extrusion of whey begins in........ days. 
Coagulum slowly peptonized, rapidly peptonized. 
Peptonization begins on..... d, complete on..... d. 
Reaction, 1d...., 2d...., 4d...., tod...-, 2od.... 
Consistency, slimy, viscid, unchanged. 

Medium browned, reddened, blued, greened. 

Lab ferment, present, absent. 


. Litmus Milk. 


Acid, alkaline, acid then alkaline, no change. 
Prompt reduction, no reduction, partial slow re- 
duction. 


. Gelatin Colonies. 


Growth, slow, rapid. 

Form, punctiform, round, irregular, ameboid, my- 
celioid, filamentous, rhizoid. 
Elevation, flat, effuse, raised, 

crateriform (liquefying). : 
Edge, entire, undulate, lobate, erose, lacerate, fimbriate, 
filamentous, floccose, curled. 


Agar Colonies. 
‘Growth slow, rapid (temperature......-- Js 
Form, punctiform, round, irregular, ameboid, my- 
celioid, filamentous, rhizoid. 
Surface smooth, rough, concentrically ringed, radiate, 
striate. 
flat, 


Elevation, 
umbonate. 

Edge, entire, undulate, lobate, erose, lacerate, fimbriate, 
floccose, curled. 

Internal structure, amorphous, finely-, coarsely~ 
granular, grumose, filamentous, floccose, curled. 

Starch Jelly. 

Growth, scanty, copious. 

Diastasic action, absent, feeble, profound. 


Medium stained 

Silicate Jelly (Fermi’s Solution.) 

Growth copious, scanty, absent. 

Medium stained..........-- 

Cohn’s Solution. 

Growth copious, scanty, absent. 

Medium fluorescent, non-fluorescent. 

Uschinsky’s Solution. 

Growth copious, scanty, absent. 

Fluid viscid, not viscid. 

Sodium Chloride in Bouillon, 

Per cent inhibiting growth 

Growth in Bouillon over Chloroform, unrestrained, 
feeble, absent. 

Nitrogen. Obtained from peptone, asparagin, glyco- 
coll, urea, ammonium salts, nitrogen. 

Best media for long-continued growth 


convex, pulvinate, 


effuse, raised, convex, pulvinate, 


19. 
III. PHYSICAL AND BIOCHEMICAL FEATURES 
1. Fermentation-tubes con- B) Blo) LE la 
taining peptone-water or $)-4| 9 £\8 8 
Sugar-free bouillon and AIRIGIENK: 
Alas Sols 


Gas production, in per cent a 


(cos) 


Growth in closed arm 

Amount of acid produced id. 
Amount of acid produced 2d. 
Amount of acid produced 4d. 


2. Ammonia production, feeble, moderate, strong, 
absent, masked by acids. 
3. Nitrates in nitrate broth, 
Reduced, not reduced. 
Presence of nitrites.........-ammomia......++++++ 
Presence of nitrates........- free nitrogen......++- 


aoa on 


Oo ow 


. Sunlight: Exposure on ice 


. Indol production, feeble, moderate, strong. 
. Toleration of Acids: Great, medium, slight. 


Acids tested 


. Toleration of NaOH: great, medium, slight. 
. Optimum reaction for growth in bouillon, stated 


in terms of Fuller’s scale.........--- 


. Vitality on culture media: brief, moderate, long. 
. Temperature relations: 


Thermal death-point (10 minutes exposure in 
nutrient broth when this is adapted to growth of 
organism)........-- Cc. 

Optimum temperature for growth........ C.: or 
best growth at 15°C., 20°C., 25°C., 30°C., 37°C., 
40°C., 50°C., 60°C. 

Maximum temperature for growth.......... Cc. 

Minimum temperature for growth.......... Cc. 


. Killed readily by drying: resistant to drying. 
. Per cent killed by freezing (salt and crushed ice or 


liquid air).......... 

in thinly sown agar 
plates: one-half plate covered (time 15 minutes), 
Sensitive, not sensitive. 

Per cent killed 


. Acids produced.........+--- 
. Alkalies produced... 
. Alcohols 
. Ferments: pepsin, trypsin, diastase, invertase, pectase, 


cytase, tyrosinase, oxidase, peroxidase, lipase, 
catalase, glucase, galactase, lab, etc.........ee sree 


17. Crystals formed:....... 0. cee ee eee eee teens 
18. Effect of germicides: 
ee Se 
lo | B Ie 
| 3/8 -38 
3 bo 
Substance Method used 2 | e ES oq 
8 | 3 wo | H's 
po i] he 
a) @)2 lq3 
2) a] [4s 
| 
: ceaee | ae te ee 
| 
=m page b 
| 
areas eae = a a — 
IV. PATHOGENICITY. 
1. Pathogenic to Animals. 

Insects, crustaceans, fishes, reptiles, birds, mice, rats, 
guinea pigs, rabbits, dogs, cats, sheep, goats, catile, 
horses, MONKEYS, MAN... 6 cece ee cere eee ee 

2. Pathogenic to Plants: 


TAN HR WwW 


. Toxins, soluble, endotoxins. 

. Non-toxin forming. 

. Immunity bactericidal. 

. Immunity non-bactericidal. 

. Loss of virulence on culture media: prompt, gradual, 


not observed in.........4-. months. 


BRIEF CHARACTERIZATION 

Mark + or O, and when two 
terms occur on a line erase the one 
which does not apply unless both 
apply. 


Diameter over Ip | 


5 Chains, filaments ey 

a ‘Endospores re 

a Capsules a 

is “Zoogloea, Pseudozoogloea, 

@ | Motie a 
| Involution forms 


Gram’s Stain 


wy | Cloudy, turbid 
3 Ring igi 
= | Pellicle 
Sediment 8 
| Shining 
> | Dull 
a 
5 & | Wrinkled 
| Chromogenic 
4 Round ae 
G Jo 5 
ra , | Proteus-like 
ke y Rhizoid 
fe = Filamentous me 
has Curled 
ey 2a) Surface-growth Fars 
os 5©| Needle-growth ase 
"S Moderate, absent 
g Abundant Ex 
= Discolored ae 


Starch destroyed 

Grows at 37°C. 

Grows in Cohn’s Sol. 
Grows in Uschinsky’s Sol. 
Gelatin (4) 

‘Blood-serum 


la 


me 


ae 
ct. 
ic} . 
58, Casein 


~oevjenb 


Agar, mannan 
Acid curd 
5 Rennet curd 


7 | Casein peptonized 
Indol (5) 
Hydrogen sulphide 


Ammonia (5) 
Nitrates reduced (5) - 


Fluorescent 


SGUNLVAd TVOINAHOOIa 


Luminous 


Animal pathogen, epizoon 
Plant pathogen, epiphyte ae 
Soil 

Milk 

Fresh water 


‘Salt water 


NOILAGIULSIA 


CHAPTER IX 


THE DISTRIBUTION OF MICRO-ORGANISMS AND 
THEIR RELATION TO SPECIAL HABITATS 


General Distribution Micro-organisms are very generally 
distributed over the surface of the earth and in its waters, and 
are carried about as dust in the air. They flourish abundantly 
in the digestive canals of animals and on their body surfaces. 
Wherever there is organic matter, the dead remains of animal 
and plant life, there are micro-organisms in abundance living 
upon the dead material and, if the temperature and moisture be 
suitable, transforming it into simpler chemical substances. In 
the soil, bacteria, yeasts, molds and-protozoa are fairly numerous, | 
especially in fertile soils near the surface. Their number rapidly 
diminishes in the deeper layers, and at a depth of six to twelve 
feet they are very scarce or entirely absent. The surface waters 
of the earth contain large numbers of bacteria and protozoa, 
especially numerous where organic matter is abundant. The air 
contains considerable numbers of molds and bacteria suspended 
as dust. The deep layers of the soil and water below impervious 
rock strata are free from micro-organisms. The surfaces of snow- 
covered mountains and of the frozen polar regions of the earth, as 
well as the atmosphere in these regions, are practically free from 
microbes. The atmosphere over large bodies of water during 
calm weather, the air in damp cellars, in sewers and in undisturbed 
rooms is germ-free, because the suspended dust particles settle 
out and do not escape again into the air unless swept up byair 
currents, which must be rather violent to remove them from moist 


surfaces. 
r8r 


182 GENERAL BIOLOGY OF MICRO-ORGANISMS 


The environment and the surfaces of growing plants and 
animals are rich in micro-organisms, especially bacteria, but the in- 
terior of the living tissues is generally germ-free in health. To this 
statement there are certain exceptions, namely, the occurrence 
of a few bacteria in thé liver, the thoracic duct and the blood of ' 
animals during active digestion, which are, however, soon de- 
stroyed by the healthy tissue; and the invasion of the root tissues 
of leguminous plants by Ps. radicicola and the growth of the bac- 
terium within the plant tissues, which results not in injury to 
the host but in a definite improvement of its nutrition by enabling 
it to utilize atmospheric nitrogen. 

Micro-organisms of the Soil——The germ-content of soil de- 
pends chiefly upon the amount of organic matter present. They 
may be present in millons per gram of soil. Bacteria, molds and 
protozoa are the most numerous. Their relation to soil fertility 
seems to be important, and they probably play a large part in 
preparing the organic matter of the soil for use as food by plants. 
A great many soil bacteria decompose protein and set free am- 
monia, and the urea bacteria are especially important in the 
transformation of urea and of animal manures into ammoniacal 
compounds. The transformation of ammoniacal compounds 
into nitrates, so-called nitrification, is accomplished by the nitri- 
fying bacteria, of which a few species have been obtained in pure 
culture, Nitrosomonas of Winogradski which produces nitrite 
from ammonia, and his genus Nitrobacter which oxidizes nitrites 
to nitrates. Very many species of soil bacteria are able to change 
nitrogen in the opposite direction, reducing nitrates to nitrites 
and further to ammonia or to free nitrogen gas. Of special inter- 
est are the soil bacteria which are able to fix atmospheric nitrogen, 
that is, absorb nitrogen from the air and combine it so as to make 
it available for plant food. The various species of the genus 
Azotobacter (A. chroococcum, A. beyerincki) accomplish this as 
they grow in the presence of dextrose, and the organism of the 
root tubercles, Pseudomonas radicicola, fixes nitrogen as it grows 
within the tissues of the legume roots. Numerous soil bacteria 


THE DISTRIBUTION OF MICRO-ORGANISMS 183 


ferment sugars, starches and fats, and there are several known 
species capable of dissolving cellulose.! 

Pathogenic Soil Bacteria——Certain pathogenic bacteria are 
of common occurrence in the soil. Whether this is their normal 
habitat or whether they gain entrance to the soil with animal 
excrement, may be questioned. At any rate the pathogenic an- 
aérobes, B. edematis, B. tetani, and B. welchii, are likely to occur 
in garden soil, and it seems probable that they actually multiply 
there to some extent. Bact. anthracis also occurs in the soil of 
fields where the disease has prevailed, and it is not improbable. 
that this organism multiplies in the ground at times. Other 
pathogenic bacteria, such as those of typhoid and cholera, seem 
to be rather quickly eliminated in the struggle for existence under 
the conditions found in surface soils. 

Micro-organisms of the Air.—Micro-organisms exist in the air 
only as floating particles of dust, or as passengers on small drop- 
lets of moist spray,.or as parasites on or in winged aérial creatures. 
Those floating as dust are derived from the earth’s surface, and 
most of the living germs usually found in this condition are the 
spores of molds. Living tubercle bacilli are unquestionably 
suspended in the air as dust, especially in the dry sweeping of 
floors where careless consumptives have lived. The spores of 
anthrax bacilli may also be suspended in the air where hides or 
wool of anthrax animals are handled. Other pathogenic bacteria 
may at times float as dust, but their presence in the air in this 
condition is apparently rather uncommon, and should be expected 
only in the fairly recent environment of cases of the disease. The 
moist droplets, expelled from the mouth and nose in speaking, 
in coughing and especially in sneezing, may remain suspended in 
the air for many minutes and be distributed to considerable dis- 
tances. After drying the solid’ material may still float as dust. 
Pathogenic micro-oganisms may readily be transmitted from 
person to person in this way. 


1 For a discussion of the microbiology of the soil, see Monograph by Lipman in 
Marshall’s Microbiology: 1920. 


184 GENERAL BIOLOGY OF MICRO-ORGANISMS 2 


In a rough way one may obtain some knowledge of the charac- 
ter of the organisms in the air of a given locality by removing the 
cover of a Petri dish containing sterilized gelatin or agar for a 
few minutes, replacing it, and allowing the organisms to develop. 
In most cases a large proportion of the growths that appear will 
be molds. Yeasts are also common, and among the bacteria 
the micrococci are abundant. Chromogenic varieties are likely 
to be present. 

A few studies of this character will show that the number 
of organisms that are present depends chiefly upon whether the 
air is quiet or has recently been disturbed by drafts, gusts of 
wind, or sweeping. ‘These facts are of fundamental importance | 
in laboratory work, if we wish to avoid contaminations. Among 
various devices that have been proposed for the accurate study 


Fic. 84.—Sedgwick-Tucker aérobioscope. 


of the organisms of the air, the Sedgwick-Tucker aérobioscope 
is the simplest and most accurate. It consists of a glass tube, 
one end of which.is drawn out so as to be smaller than the other. 
The small end contains a. quantity of fine granulated sugar; 
both ends are plugged with cotton, and the instrument is sterilized. 
A definite quantity of air is to be aspirated through the large 
end, after removing the cotton, and this may be done by means 
of a suction-pump applied to the other end, or by siphoning water 
out of a bottle, the upper part of which is connected with the end 
of the aérobioscope by means of a rubber tube. The sugar acts 
as a filter and sifts out of the air the micro-organisms which are 
contained in it. Liquefied gelatin or agar may be introduced 
into the large end of the instrument by means of a bent funnel; 
and, after replacing the cotton, it is mixed with the sugar which 
dissolves. The culture-medium may be spread around the inside 
of the larger portion of the tube after the manner of an Esmarch 


THE DISTRIBUTION OF MICRO-ORGANISMS 185 


roll-tube. The microbes which are filtered out by the sugar will 
develop as so many colonies upon the solidified medium. 

Many important micro-organisms, and certainly some germs 
of disease, are borne through the air by the winged insects, and 
to a less extent by birds. The microbes are found not only on 
the feet and outer body surfaces of these carriers, but they also 
occur on and in the mouth parts, in the alimentary canal and 
sometimes in the interior of the animal’s body tissues. Certain 
pathogenic micro-organisms (Plasmodium, Trypanosoma) are 
known to be transmitted from one person to another almost 
exclusively by biting insects, and the importance of these carriers 
in air-borne disease of both animals and plants, is being recognized 
more and more. 

Micro-organisms of Water and Ice.—The water of rivers, 
lakes and the ocean always contains bacteria. The number 
or organisms varies greatly in different places and under different 
conditions. The number of different species found in water 
is also very large. Some of these, the natural water bacteria, 
including many bacilli which produce pigment and some cocci 
and spirilla, seem to live in surface water as their natural habitat. 
With the addition of putrescible material these forms are in- 

creased in number and certain of them (Proteus group, fluorescing 
~ bacteria) become numerous. Soil bacteria are numerous in 
waters during floods and after rain, and they may persist for 
some time. Intestinal bacteria occur in waters which receive 
sewage or are otherwise contaminated with excreta. They 
persist only a relatively short time. Certain intestinal protozoa, 
Endameba, Balantidium, seem also to occur in waters at times. 
Ground-water! contains few or no bacteria under normal condi- 
tions, and is therefore suitable for a source of water-supply, 
-when a sufficient amount is available. The possibility of contami- 
nation of the ground-water from unusual or abnormal conditions 


1 Ground-water is the water which—originally derived from rain or snow—sinks 
through superficial porous strata, like gravel, and collects on some underlying, 
impervious bed of clay or rock. 


186 GENERAL BIOLOGY OF MICRO-ORGANISMS 


should always be eliminated before it is taken for drinking 
water. Numerous epidemics of typhoid fever have been traced 
to contamination of wells. The location of wells with reference 
to privy-vaults and other possible sources of contamination 
should be chosen with the greatest care. 

The ordinary bacteria of water are harmless, as far as is 
known.! Bad‘odors and tastes in drinking water that is not 
polluted with putrid material. are usually due to minute green 
plants (alge).2 The diseases most commonly disseminated. 
by water are typhoid fever and Asiatic cholera, and probably 
also dysentery. The spirillum of cholera will usually die in 
natural water (not sterilized water) inside of two or three weeks; 
the bacillus of typhoid fever will usually die in two or three weeks. 
Under exceptional circumstances these organisms may perhaps 
maintain their vitality for a longer period. They appear, however, 
to be less hardy than the ordinary water bacteria. As we now 
understand these diseases, the organisms causing them will be 
present only in a water-supply which has been recently con- 
taminated by the excreta from a case of the disease. Notwith- 
standing the rapid death of these organisms in water, they may 
exist long enough to infect individuals habitually drinking the 
water. Many epidemics of cholera and typhoid fever have been 
traced to water polluted with the discharges from’ cases of these ‘ 
diseases, and in a few instances the relation of the contaminated 
water supply to the epidemic has been established beyond a 
reasonable doubt. 

By self-purification of water is meant the removal, through 
natural processes, of contaminating organisms such as might 
occur from the discharge of sewage into it. It depends upon 
the sedimentation of the contaminating material in the form 
of mud, upon the growth of the ordinary water-plants and protozoa, 


1See Fuller and Johnson, ‘‘The Classification of Water Bacteria,” Journal 0 
Experimental Medicine, Vol. IV, p. 609. 

2“Contamination of Water Supplies by Alge.”’ G. T. Moore in Yearbook 
U.S. Department of Agriculture, 1902. 


THE DISTRIBUTION OF MICRO-ORGANISMS 187 


upon the exhaustion of the food supply by the growth of bacteria 
themselves, upon the destructive influence of direct sunlight, 
and the dilution of the contamination by a large volume of 
water.’ It is not usually to be relied upon as a means of freeing 
the water-supply from pathogenic bacteria. 

Storage of Water—When water is kept in large reservoirs, 
the solid particles in it, including bacteria, tend to fall to the 
bottom. The number of bacteria in a water-supply may be 
considerably reduced in this way. The use of large storage 
reservoirs also provides for the dilution of any sudden excess of 
pollution, and if the water is held in storage the pathogenic 
germs present disappear for the most part in a few days or weeks. 

Filtration—Water may be completely sterilized by passing 
it through the Pasteur-Chamberland filters of unglazed porcelain, 
or through the more porous Berkefeld filters. Such filters are 
effective only when frequently cleaned and baked, and in practical 
purification of water for household purposes they usually fail 
because of the intelligent care they require. Other types of 
domestic filters are generally worse than useless. 

Filtration on a large scale is commonly employed in the purifi- 
cation of water supplies of cities. By this method 98 per cent to 
99 per cent of the bacteria in water may be removed. 

Slow Sand Filtration.’—The filter consists of successive layers 
of stones, coarse and fine gravel. The uppermost layers are 
of fine sand. The whole filter is from 1 to 2 meters thick. The 
sand should be 60 cm. to 1.2 meters in thickness. The accumu- 
lated deposit from the water and a little of the fine sand must 
be removed from time to time, but the layer of fine sand must 
never be allowed to become less than 30 cm. in thickness. The 
first water coming from the filter is discarded. The actual fil- 
tration is done largely by the slimy sediment which collects 
on the surface of the layer of fine sand. The filterbeds may 


1See Jordan, Journal of Experimental Medicine. Vol. V, p.:271. 
2 For a full discussion see Journal American Medical Association. Oct. 3 to 


31, 1903. 


188 GENERAL BIOLOGY OF MICRO-ORGANISMS 


-be several acres in extent, and in cold climates should be pro- 
tected by arches of brick or stone. They require renewal occa- 
sionally. This kind of filtration has come largely into use since 
the cholera epidemic of 1892-93, and it appears to be very effective. 
It is important to use storage basins in connection with sand 
filtration. 

The results obtained by filtration depend greatly upon the 
intelligence displayed’ in operation, and must be controlled by 
frequent examinations of the water. 

Mechanical Filtration—This method of filtration is also 
called the American system. It is more rapid than the preceding 
method and does not require a large area-for filter’ beds. Al- 
though sand is required also, filtration is accomplished by a 
jelly-like layer of aluminium hydroxide. This product is formed 
by adding to the water a small quantity of aluminium sulphate 
or of alum. The carbonates in the water decompose the alumin- 
ium salt and produce aluminium hydroxide. It precipitates as a 
white, flocculent deposit, entangling solid particles, including 
bacteria, as coffee is cleared with white of egg. Only a trace 
_ of aluminium should appear in the water. This methed of filtra- 
tion has not been tested so extensively as slow sand filtration, 
but seems likely to proveefficient. With water poor in carbonates, 
these may have to be added.* 

Whipple and Longley? found that the efficacy of mechanical 
filters with the addition of alum depends somewhat upon the 
character of the alum. They find that the alum shall be shown 
by analysis to contain 17 per cent of alumina (Al,O3) soluble 
in water, and of this amount at least 5 per cent shall be in excess 
of the amount necessary theoretically to combine with the sul- 
phuric acid present. It shall not contain more than 1 per cent 
of insoluble substances, and shall be free from extraneous debris 
of all kinds. It must not contain more than o.5 per cent of iron 
(Fe,O3) and the-iron shall be preferably in the ferrous state. 


1See Fuller, Journal American Medical Association, Oct. 31, 1903. 
2 Journ. Infect. Diseases, Supplement No. 2, Feb., 1906, pp. 166-171. 


THE DISTRIBUTION OF MICRO-ORGANISMS 189 


Chemical Disinfection—Various methods for the purification 
of water by means of chemicals have been proposed. The use 
of copper sulphate to disinfect drinking water was recommended 
by Moore and Kellerman,} and various investigators tested the 
value of their recommendation. Clark and Gage? came to the 
conclusion from their investigation that the treatment of water 
with copper sulphate or the storing of water in copper vessels 
has little practical value. Others also have come to practically 
‘the same conclusion. While the addition of copper sulphate is 
of use in preventing the growth of the alge, which sometimes 
grow so abundantly as to choke up water pipes, and is of benefit 
in this direction, the weight of evidence appears to be against 
its efficacy for purifying water for drinking purposes. More 
effective chemical disinfection has been obtained by means of 
ozone generated by electricity. More recently, calcium hy- 
pochlorite and free chlorine have been employed for this purpose 
with considerable success, and have almost completely displaced 
other substances as chemical disinfectants of drinking water. 

Physical Disinfection—The most effective and surest method 
of disinfecting drinking water is by boiling it or by distillation. 

Bacteriological Examination of Water—For bacteriological 
examination samples from the water-supply of a city may be 
drawn from the faucet, but the water should first be allowed to 
run for half an hour or longer. From other sources the supply 
should be collected in sterilized tubes or bottles, taking care to 
avoid contamination. These samples should be examined as 
promptly as possible, for the water bacteria increase rapidly 
in number after the samples have been collected. When trans- 
portation to some distance is unavoidable the samples should 
be packed in ice, but even this precaution does not preserve the 
original bacteriological condition of the water at the time of 
collection; for more or less change probably takes place at all 
temperatures. If the temperature is too low, and the water 


1U. S. Dep. Agriculture, Bu. Plant Ind. Bulletin 64, 1904. 
2 Journ. Inf. Diseases, Sup. No. z, Feb., 1906, pp. 175-204. 


196 GENERAL BIOLOGY OF MICRO-ORGANISMS 


MICRO-ORGANISMS OF Foop : 
Milk.— Milk is the natural food of young mammals, and 
naturally it is taken directly from the mammary gland into 
the digestive tract of the young mammal. For many centuries 
however, the milk of certain animals has been extensively used 
as a commercial food forman. The principal animals furnishing 
commercial milk are the cow, goat and mare. The chemical 
composition of milk is different in different animals, in the same 
animal at different periods of lactation, and even that obtained 
at different stages of a single milking shows considerable varia- 
tion. In general cow’s milk has the following composition. 


Variation ~ Average 
aE si caxseccanen sins each Gules taweniee a eer 3-6 4 per cent. 
AGES Eee cst aucassesopeg uch west Sap a sees seea nce 1-3 2 per cent. 
Protein...7........ Saye Sia ery git oneal 5-8 7 per cent. 
Water sunisovas + conf AS ka GE Se eR Ee 84-88 87 per cent. 


It is an excellent medium for the growth of most bacteria and is 
commonly used in the laboratory for this purpose. 

There are about 200 species and varieties of bacteria which 
commonly occur in milk. They are derived in part from the 
udder itself. Bacteria are always present in the milk ducts of 
the udder and are fairly abundant in the first portions of milk 
drawn, so that milk very carefully drawn from healthy animals 
may contain 200 to 400 bacteria per cubic centimeter. Milk 
from diseased udders may be very rich in pathogenic micro- 
organisms. As the milk is drawn, many micro-organisms usually 
gain entrance to it from the atmosphere, the hands of the milker 
and the utensils with which it comes in contact. From the 
body of the cow, particles of dust and hairs drop into the milk, 
carrying with them the flora of the intestine and of the skin of 
thecow. From the milker, the material on the hands and possibly 
also from the nose and mouth may reachthe milk. The utensils, 
unless sterilized before use, contribute the microbic flora of the 
previous milkings, of the water used for cleansing and from the 


THE DISTRIBUTION OF MICRO-ORGANISMS 197 


person who handles them. From the air, the milk may receive 
further contamination (1) from flies coming to drink or perhaps 
to drown without a clean bill of health from their port of last 
departure, (2) from particles suspended as dust and containing 
micro-organisms derived from manure, from hay and straw, 
and from soil, and (3) moist droplets expelled from the mouth and 
nose of the milkers and of the cattle. The subsequent hand- 
ling of the milk may add further kinds of bacteria from human 
sources. Modern dairy practice in vogue in the production 
of the higher grades of milk eliminates some of these sources 
of contamination and minimizes the importance of the rest, but 
nevertheless fresh milk of even the better grades contains a 
great variety of micro-organisms, and often as many as 10,000 
to 100,000 per cubic centimeter when it leaves the producer’s 
dairy. 

The usual milk flora derived from these various sources may 
be classed under the following heads: 

A. Lactic acid bacteria. 

1. Bacterium (streptococcus?) acidi lactici 
2. Bacillus coli and B. lactis aérogenes. 
3. Long rods of B. bulgaricus type. 

4. Streptococcus pyogenes. 

5. Micrococcus acidi lactict. 

6. Acid formers which liquefy gelatin. 

B. Gelatin-liquefying bacilli. 

7. Rapidly liquefying types—B. subtilis. ° 

8. Slowly liquefying types. 

C. Pigment-forming bacteria. 

D. Anaérobic bacteria—B. welchii, putrefactive anaérobes. 

E. Special types causing peculiar fermentations, such as 
slimy consistency, bitter taste and peculiar odors. 

F. Pathogenic organisms—typhoid, tuberculosis, scarlatina, 
diphtheria, diarrhea, septic sore throat, foot-and-mouth disease, 
dysentery. 

G. Other fungi—Molds, Oidia, Yeasts, Actinomyces. 


198 GENERAL BIOLOGY OF MICRO-ORGANISMS 


‘The development of these various microbes in the milk de- 
pends very much upon the temperature at which it is kept. At 
o° to 10° C. the acid-forming bacteria grow very slowly or not 
at all, and the milk may remain practically unchanged for many 
days or even weeks. Eventually some of the liquefying bacilli 
or the slime-producing types may gain the upper hand and change 
the consistency and flavor. Between 10° and 21° the Bact. acidi 
lactict is almost certain to gain the dominance and rapidly to 
suppress the other types, and it produces the normal souring of 
milk. Between 21° and 35° C. the organisms of the B. colt and 
B. lactis aérogenes groups are likely to predominate and at tempera- 
tures from 37° C. to 40° C. the B. bulgaricus is likely to gain the. 
ascendency, after a few daysat any rate. These may be regarded 
as the normal fermentations of unheated milk of very good 
quality. The other microbes in the milk are not destroyed by 
these fermentations but their development is usually held in 
check somewhat. 

Shortly after the coagulation of the milk, which occurs when 
the lactic acid reaches a concentration of about 0.45 per cent, 
the living bacteria begin to diminish in number, and gradually 
Oidium lactis and other molds become prominent, although acid- 
resisting forms such as B. bulgaricus still continue to grow. 
Organisms of these kinds seem to be specially concerned in the 
ripening of acid curd in cheese making. Finally the acidity may 
disappear as a result of the activity of molds, and putrefactive 
bacteria find the opportunity to develop. 

If the milk be pasteurized, the bacteria which form lactic 
acid are killed, and when fermentation occurs it is likely to be 
different from the normal souring. ‘At a high temperature, 
the stormy butyric-acid fermentation due to B. welchizt may be 
observed. At a lower temperature, a slow putrefaction due to 
spore-forming putrefactive anaérobes in conjunction with other 
bacteria may occur. These fermentations are ordinarily inhib- 
ited by the lactic acid produced in the normal souring of milk. 

Alcoholic fermentation of milk occurs as a rule only when 


THE DISTRIBUTION OF MICRO-ORGANISMS 1gQ 


special ferments are purposely added to produce this result. 
Kumyss and Kefir are fermented milks produced in this way. 
The starter or ferment contains yeasts as well as bacteria. 

The pathogenic micro-organisms in milk are derived in part 
from unhealthy cows—tuberculosis, foot-and-mouth disease, 
septic sore throat (?)—but in a larger measure from the people 
who handle the milk or from utensils—tuberculosis, typhoid 
fever, scarlatina, diphtheria, diarrheas, dysentery, septic sore 
throat (?). The bacteria of typhoid fever, diphtheria and dysen- 
tery are known to multiply in milk. The microbes of tuberculo- 
sis and foot-and-mouth disease may persist in butter and cheese 
for several weeks at least. 

Leaving out of consideration the question of specific patho- 
genic micro-organisms; the presence of more than 500,000 bac- 
teria per cubic centimeter in the milk regularly fed to infants 
and young childern is undoubtedly harmful, and especially so 
in warm weather. Doubtless many factors contribute to the 
causation of the summer diarrheas and the summer mortality 
of children, but there can no longer be any question that a milk 
rich in living bacteria as food for these children is one of the very 
important causes of their illness and death. 

Milk for infant feeding should come from clean, healthy 
(tuberculin-tested) cows, should be handled by clean healthy 
workmen, in clean stables and rooms and with clean, sterilized 
utensils. It should be bottled at the producing dairy, promptly 
chilled to 10° C. or below, and maintained at this temperature 
until delivered at the home. At this time the living bacteria 
should not exceed 30,000 per cubic centimeter. In the home, 
the milk should be kept cold. It must be handled only with 
utensils sterilized by boiling in water. Boiled water is employed 
in making the necessary dilutions. If the milk supply is not 
above suspicion the milk should be pasteurized by heating to 
60° C. for 20 minutes. The dilution is prepared and filled into 
separate bottles sufficient in number so that one may be used 
for each feeding during the succeeding 24 hours. Each bottle 


200 GENERAL BIOLOGY OF MICRO-ORGANISMS 


is chilled in cool water, then ice water, and finally stored in the 
refrigerator. Immediately before feeding it is warmed by partial | 
immersion in warm water. 

Other Foods.—Other foods, meats, fish, eggs, vegetables 
and fruits, undergo decompositions due to more or less definite 
types of micro-organisms, and the activities of these are delayed 
or prevented by modern methods of preserving foods, in some 
instances very successfully, and in other cases with limited 
success.1 Any food, and especially that eaten without cooking, 
may serve as a passive carrier of pathogenic micro-organisms. 
Salads, green vegetables and fresh fruits may undoubtedly act in 
this way during epidemics. Oysters taken from sewage-polluted 
beds have been found to convey typhoid fever. Meats derived 
from mammals may contain specific germs causing disease in 
both animals and man, such as tuberculosis, anthrax and foot- 
and-mouth disease.- The flesh of bovine animals suffering with 
enteritis at the time of slaughter seems to be particularly liable 
to develop poisonous properties, and the ill effects observed in 
these instances may have been due to a specific infection. Para- 
typhoid fever is sometimes traced to such meat as a cause. 

Meats and fish are rich in protein and their decomposition 
by saprophytic bacteria may give rise to various poisonous sub- 
stances, as has been mentioned on page 177. The usual course 
of putrefaction, however, goes on without very strong poisons | 
being produced, as we may judge from the habitual use of partly 
decomposed foods of this sort. Virulent poisons are occasionally 
encountered and some of these are due to the presence of specific 
microbes, B. botulinus of Van Ermengen, B. enteritidis of Gaertner 
and the paratyphoid and paracolon bacilli. 


1 For a discussion of the microbiology of foods and of food preservation see 
Marshall’s Microbiology for agricultural and domestic science students, 1920. 


CHAPTER X 
PARASITISM AND PATHOGENESIS 


The Parasitic Relation.—The presence in a living organism 
of one or several organisms of another species, which live as para- 
sites upon the first, is a phenomenon of common occurrence in na- 
ture. Those organisms such as the bacteria, which are too small 
to harbor visible internal parasites, are subject to the parasitic 
ravages of larger beings such as amebe and other protozoa, 
which engulf them bodily and digest them. Man, who is wont 
to complain of his parasitic ailments, takes all his protein, fat 
and carbohydrate from the bodies of plants and other animals. 
Parasitism in the larger sense is a well-nigh universal character- 
istic of living beings. Parasitism in a narrower sense usually 
applies to the existence of a smaller organism, the parasite, in 
or on the body of a larger, the host, a relation in which the host 
furnishes the parasite its necessary food. In many instances the 
advantages of the relation are wholly one-sided, but in others 
the two organisms seem to be of mutual benefit. In the latter 
case, the condition is called symbiosis. The infection of the 
roots of the clover with Pseudomonas radicicola, which promotes 
the nitrogenous nutrition of the plant, is an example of this rela- 
tion. In other instances the two organisms living in close associa- 
tion seem neither to help nor injure each other. They are then 
called commensals or companions at the same table. Internal 
parasites occur in all the higher animals and plants, and have 
been found even in the bodies of protozoa. Representatives of 
all the great classes of micro-organisms are found among the 
internal parasites, and many more highly organized animals 


and plants also lead parasitic lives. Man, alone, is subject to 
201 


202 GENERAL BIOLOGY OF MICRO-ORGANISMS 


infestation with parasitic insects and numerous worms, in addi- 
tion to an enormous variety of microbes. Whether a parasitic 
‘organism is to be regarded as a symbiont, a commensal or a 
pathogenic agent depends upon the effect which it produces 
upon its host. A pathogenic organism is one whose presence 
results in definite injury to the host. 

Pathogenesis.—In human pathology the phenomena of dis- 
ease have for centuries been the object of careful study and 
speculation, and in many instances the phenomena commonly 
associated together have long been regarded as a complex result 
of a single primary cause, and the condition in which such phe- 
nomena are observed has been regarded as a single morbid en- 
tity or a definite disease. Even the most ancient‘records indicate 
that such recognition had long’ been common knowledge. A 
beginner in parasitology or pathology may be inclined to ascribe 
a causal relation to a parasite which he observes in the body of a 
sick individual; in. fact this has been done repeatedly. The log- 
ical requirements for the proof of such a relationship were first 
formulated by Henle, as has been mentioned in the historical 
sketch in the introductory chapter. They were reformulated 
by Koch, who, for the first time, was able to comply with them 
in respect to a bacterial disease. They may be stated as follows: 


1. The organism must be present in all cases of the particular 
disease. ; 

2. The organism must be isolated from the diseased body 
and propagated in pure culture. 

3. The pure culture of the organism when introduced into 
susceptible animals must produce the disease. 

4. In the disease thus produced, the organism must be found 
distributed as in the natural disease. 


Although we may very properly consider a micro-organism as 
the probable cause of a disease with which it is associated, with-~ 
out satisfying all of the above requirements, experience has 
served to emphasize more and more the wisdom of reserving final 


PARASITISM AND PATHOGENESIS 203 


judgment wherever these rules or similar stern logical requirements 
have not been satisfied. 

‘Infectious Disease.—An inféctious disease is a disease due 
to the entrance of a living micro-organism and its growth in the 
body. Although conservative bacteriologists are sometimes loth 
to accept a disease as infectious until Koch’s rules have been 
satisfied, most are agreed that a disease, which can be reproduced 
indefinitely by the inoculation of healthy individuals in series with 
material taken from a preceding case, is due to a living cause. 
The proof that a disease is due to a living cause may therefore 
precede the identification of the causal organism, often by many 
years. 

Possibility of Infection—Whether a parasitic organism will 
be able to enter and multiply in a new host and cause disease 
depends upon a number of circumstances, the most important 
of which may be considered under four heads, namely, the quality 
of.the microbe, the resistance of the host, the quantity of invading 
parasites, and the path of entrance. The course and ultimate 
result of an infection depend also to a marked degree upon these 
same factors. In general the qualifications of the micro-organism 
depend first upon the experience of its ancestry under the same 
or similar environmental conditions, factors inherent in its species, 
and second, upon its very recent history, factors affecting the 
virulence and general vigor of the individual microbe. Thus 
the tubercle bacillus is qualified by inheritance for a parasitic 
existence, while the common yeast cell is not. Yet, the tubercle 
bacillus, when cultivated for a long time on artificial media may 
lose its former ability to grow in the animal body. The factors 
affecting the pathogenic properties of a microbe will be considered 
in the succeeding chapter. 

Susceptibility and Resistance.—Among the important things 
in the nature and condition of the host, we need also to consider 
both racial and individual characters. Certain species of animals 
have harbored certain parasites for so long that the latter have 
become adapted to growth in the particular species of host. “In 


204 GENERAL BIOLOGY OF MICRO-ORGANISMS 


some instances the adaption is very narrow and the parasite 
may be able to exist naturally only in the one-host species, as for 
example Spirocheta pallida. Individual resistance of different 
hosts of the same species is variable. Age is one important fac- 
tor: there are the children’s diseases, measles, chickenpox; the 
diseases of active adult life, pulmonary tuberculosis, typhoid 
fever; and the diseases of the aged, pneumonia, carcinoma. Hun- 
ger and thirst have been shown experimentally to reduce the 
resistance to infection: pigeons, which are normally immune to 
anthrax become susceptible when starved. The effect of fatigue 
is well known: a white-rat, normally immune to anthrax, suc- 
cumbs to it after prolonged work in the treadmill. Abnormal 
chilling of hens removes their immunity’ to anthrax and abnormal 
heating of frogs affect them in a similar way. _Chemical poisoning 
also increases susceptibility to infection, and cachexia and mal- 
nutrition are well-known predisposing factors to such infections 
as tuberculosis. Traumatism is very important, not only for its 
general effect upon the resistance of the host, but especiallyin the 
reduction of local resistance by destruction or injury of tissue 
(wounds). There are certain locations where resistance to in- 
fection is naturally lower, such as the ends of growing bones and 
the interior of the parturient uterus. 

Number of Invaders.—The quantity of infectious material 
introduced is of importance in determining whether infection 
will or will not occur. Very few species of microbes are capable 
of causing disease when only a single individual organism is in- 
troduced into the body. A large number of microhes entering 
at the same time seems to overburden the defensive powers of 
the body so that some of the parasites succeed in establishing 
themselves and multiplying. 

Modes of Introduction.—There are various avenues: by which 
micro-organisms may enter the body to produce disease. In- 
fection of the ovum in the ovary with spirochetes and protozoa 
is known to occur in some insects, and Rettger has shown that 
this phenomenon occurs in the hen infected with Bacterium pul- 


PARASITISM AND PATHOGENESIS 205 


lorum. The human ovum also seems occasionally to be infected 
with Spirocketa pallida in this way. It may also become in- 
fected with the same organism derived from the seminal fluid. 
The developing fetus is sometimes invaded by pathogenic micro- 
organisms introduced through the placental circulation. The 
organisms of tuberculosis, small-pox, typhoid fever and the 
pyogenic cocci are known to be transmitted, somewhat uncom- 
monly to be sure, in this way. Asa rule the germ must be circu- 
lating in the blood of the mother in considerable numbers, or 
there must be actual infectious lesions of the placenta before 
placental transmission occurs. After birth non-pathogenic mi- 
crobes gain access to the entire surface of the body and penetrate 
the various canals opening to the exterior to certain normal 
limits. Pathogenic germs may be introduced with the food and 
drink, which is the common natural mode of infection with cholera 
and typhoid fever in man and with tuberculosis in hogs and cattle. 
The barrier presented by the activity of the gastric juice is fre- 
quently passed in safety by the ingestedmicrobes. Inhalation 
is probably the most common way in which tuberculous infection! 
reaches the lungs in man, although there is conclusive evidence 
that tuberculosis in this location may be derived from the alimen- 
tary tract through the blood stream. Experimentally, guinea- 
pigs are much more susceptible to infection with tubercle bacilli 
by inhalation than by ingestion. Mere application of the in- 
fectious agents to the epithelial surface of the skin or mucous 
membranes results in infection in many instances and, indeed, 
infection by ingestion and inhalation may be regarded as examples 
of this. The mucous membranes of the urethra and the eye, and 
also of the rectum in young children, are especially susceptible to 
infection with the gonococcus. The unbroken skin may be in- 
fected with staphylococci, which seem to penetrate through the 
hair follicles and sebaceous glands, giving rise to boils and car- 


1 McFadyean, Journal Royal Institute of Public Health, 190, Vol. XVIII, pp. 
793-724. 


206 GENERAL BIOLOGY OF MICRO-ORGANISMS 


< 


buncles; but to most microbes the uninjured skin presents an 
effective barrier. 

The question whether infectious agents may penetrate epithe- 
lium and gain the lymph or blood-vessels beyond withcut causing 
a local lesion, has received considerable attention and it seems 
to. be established as certainly possible in the intestine during 
the absorption of fat, and it may perhaps occur in other locations. 

Infection through wounds, even minute breaks in the epithe- 
lial covering, is very common. Such wounds made by insects 
are the common portals of entry for the germs of malaria, plague, 
yellow fever, relapsing fever and many more diseases. Larger 
wounds nearly always become infected with pyogenic cocci 
unless they are properly cared for. The introduction of infectious 
material into the subcutaneous tissue may occur accidentally in 
deep wounds and is a common mode of inoculation in the labora- 
tory. Infection with the anaérobic bacillus of tetanus frequently 
occurs in this type of wound. 

Infections of the peritoneal cavity, pleural cavities and cavi- 
ties of the joints result from penetrating wounds, by the entrance 
of bacteria from contiguous tissues, as through the intestinal 
wall into the peritoneal cavity, and through the blood and lymph 
channels. 

Local Susceptibility—-The invading parasite is favored by 
conditions of local susceptibility such as tissue destruction, 
presence of necrotic tissue and foreign bodies, and also by the 
presence of other infectious microbes. Small-pox and staphylo- 
coccus, tetanus and the pus cocci, scarlet fever and streptococcus, 
are common examples of such mixed infections. In some in- 
stances one infection predisposes to another. For example, 
measles is likely to favor the development of tuberculosis; the 
caseous tubercle is likely to be invaded by the streptococcus. 
These subsequent invasions are spoken of as secondary infections. 

Local and General Infections.—The invading microbes may 
remain localized near the point of entrance, as for example in 
tetanus and diphtheria. In such cases the general effects may he 


S. 


PARASITISM AND PATHOGENESIS 207 


due to disturbance in function of the local tissue, such as laryngeal 
obstruction in diphtheria, or to the absorption into the lymph 
and blood of poisons produced at the infected site. Such ab- 
sorption results in toxemia with symptoms due to poisoning of 
distant tissue elements. On the other hand, the infectious 
agent may pass quickly to the blood stream without appreciable 
local reaction and multiply there, as in malaria, trypanosomiasis 
and streptococcus bacteremia. Again there may first develop 
an intense local reaction, with subsequent extension to the 
blood stream with fatal issue, as in malignant pustule (anthrax). 
In other instances repeated temporary invasions of the blood 
occur, with numerous localized abscesses in’ various parts of 
the body, a condition to which the name pyemia has been applied. 
Of particular interest are those general infections-of the blood 
stream, which finally fade away, but leave behind localized 
_ infections in the joints, on the heart valves, in the central nervous 
system, or other parts of the body. Sleeping sickness, syphilis, 
acute articular rheumatism and generalized gonococcus infection 
belong in this category. 

Transmission of Infection.—The manner in which an infectious 
agent passes from its host to a new victim varies considerably. 
In general it may be said to occur (1) by direct contact or close 
association, transmission by contagion, (2) through the agency of 
intermediate dead objects as passive carriers, transmission by 
fomites, or (3) through the agency of a living or dead object, in 
which the parasite undergoes development or multiplication, 
transmission by miasm. These terms have been employed in 
the past to designate rather hypothetical objects not to say 
abstract ideas, and their application to the facts learned by 
modern research is, perhaps, not desirable. Nevertheless, they 
may be made to fit the observed phenomena in a way. Thus, 
syphilis and gonorrhea are transmitted by contagion; diphtheria 
and small-pox by contagion and by fomites, tetanus and anthrax 
by fomites and perhaps also miasm, plague by contagion, fomites 
and miasm (through the rat and flea); malaria, trypanosomiasis 


208 GENERAL BIOLOGY OF MICRO-ORGANISMS 


and yellow fever by miasm. All of these are doubtless infectious 
diseases but some of them are not naturally spread by contact at 
all. In ‘studying each disease it will be necessary to consider 
the avenues by which the parasite leaves the patient, its existence 
in the external world and the means of gaining access to its new 
victim. 

Healthy Carriers of Infection—A person or animal may 
harbor virulent infectious agents without showing symptoms of 
disease, and may serve as a source of infection to others. This 
was clearly recognized in the sixteenth century by Fracastorius 
as a factor in the spread of syphilis. Only recently has its im- 
portance in other diseases been emphasized. 


CHAPTER XI 
THE PATHOGENIC PROPERTY OF MICRO-ORGANISMS 


Adaptation of Parasitism.—In order to live as a parasite, an 
organism must be adapted. to grow under the conditions met 
with in the body of the host; but in order to produce disease it 
must also injure the host. The most perfect adaptation of 
parasitism is probably exhibited by those micro-organisms 
which do not injure the host, the symbionts and commensals, 
as it is obviously to the interest of the parasite to keep its host 
alive. An. adaptation of this kind usually requires that: the 
microbe shall either grow very slowly, or shall be so situated 
that the excessive numbers resulting from its multiplication 
may readily pass out of the host or be disposed of in some way; 
otherwise the host would be physically crowded out. This sort 
of adaptation is illustrated by the normal intestinal bacteria. 
Parasites which invade the tissues of the body rarely show such 
adaptation.. It is, perhaps, approached to some extent by 
the slow-growing bacilli of leprosy and tuberculosis. In most 
instances of parasitism, however, there is more or less of a struggle 
between the invader and the host for the possession of the field, 
and the phenomena of disease are incident to this combat. 

Virulence.—The ability of the parasite to injure its host, is 
designated as virulence. Virulence depends in part upon growth 
vigor, but also upon other factors largely unknown. A great 
deal is known about specific methods of changing the virulence 
of micro-organisms, and various procedures are commonly em- 
ployed with this object in view. A diminution in virulence is 
called attenuation and an increase in virulence, exaltation. 
Attenuation was first observed by Pasteur in a culture of Bacterium 


avisepticum (chicken cholera) grown in broth in the presence 
14 209 


210 GENERAL BIOLOGY OF MICRO-ORGANISMS 


of air. Pneumococci and streptococci also attenuate rapidly in 
artificial ;culture. Even those bacteria which retain their viru- | 
lence in ordinary cultures become attenuated when grown at 
unusually high temperatures (42° C.) or in the presence of anti- 
septics, both of which methods have been employed in attenuat- 
ing the anthrax bacillus. Attenuation also results sometimes 
from parasitism in hosts of another species. Wariola and vaccinia 
present a conspicuous example of this. Mere dessication of a 
virus. seems to attenuate it in some instances (rabies) but this 
is somewhat doubtful. Many pathogenic agents become some- 
what attenuated upon long residence in the same host in chronic 
infections. Exaltation of a virus, on the other hand, is accom- 
plished by rapid passage through susceptible animals in series. 
When the organism is too attenuated to produce an infection 
alone, it may ke aided by the admixture of other organisms | 
(mixed infection) or by the presence of irritating foreign bodies 
(splinters, stone dust) or by mechanical protection in collodion 
capsules. 

Microbic Poisons.—The weapons which the pathogenic 
agent employs to injure its host are various. The physical 
mass of the invaders may be injurious, more particularly by 
obstructing blood-vessels, as in estivo-autumnal malaria in man 
and anthrax in the mouse. Usually, however, the offensive 
weapons are chiefly chemical poisons. The soluble toxins, or 
true toxins are substances. of unknown chemical composition, 
produced inside bacterial cells and passed out to their surround- 
ings. These so-called extracellular toxins include the most 
poisonous substances known. Brieger and Cohn obtained: a 
toxin, still impure, from tetanus bacilli, of which five one hundred 
millionths of a gram (.cocc0005 gram) killed a mouse weighing 
15 grams. At this rate .coo23 of a gram would kill a man weigh- 
ing 70 Kilos. The soluble toxins elaborated by the diphtheria 
and tetanus bacilli have been studied most, and many of our 
ideas concerning toxins in general have been derived from these 
studies. These poisons are rapidly destroyed by heat, resembling 


PATHOGENIC PROPERTY OF MI€RO-ORGANISMS 211 


enzymes in this respect. They differ from enzymes in that 
they are used up in combining with tissue. Thus tetanus toxin 
may be completely neutralized by the addition of brain tissue, 
and either diphtheria or tetanus antitoxin may be quantitatively 
neutralized by its specific antitoxin. Ehrlich in his study of the 
reactions of diphtheria toxin showed that on standing it loses 
much of its poisonous property without any diminution in its 
ability to combine with diphtheria antitoxin, and to this less 
poisonous substance he gave the name toxoid. From this observa- 
tion he concluded that the toxin molecule contains at least two 
very definite atomic groups. One of these is comparatively 
stable and serves for attachment of the toxin molecule to the 
cell attacked by it, and is called the haptophorous group or 
simply haptophore. The other recognizable chemical group 
disintegrates more readily and is that which bears the poisonous 
property. To this he gave the name of toxophorous group or 
toxophore. In their reactions toxins behave in part like feebly 
dissociated chemical compounds, as has been shown by Arrhenius 
and Madsen, but the reactions by which they combine are only 
slightly or not at all reversible and, moreover, take place in 
variable proportions. Bordet very aptly compares the reactions 
of toxin to the union of a dye with a stainable material. Bacteria 
also produce poisons which are part of their own body substance, 
and set free only upon their death and disintegration. These 
are spoken of as intracellular toxins. Injurious substances may 
also be produced from the tissue of the host by a secondary action 
outside the cell of the parasite, but these secondary products 
play a very minor réle. 

Defensive Mechanisms.—The defensive armor of parasites 
seems also to be in part physical and in part chemical, and perhaps 
we may regard the physiological adaptation to slow growth as a 
defensive mechanism because it tends to avoid exciting the 
opposition of the host. The physical structure seems to be 
protective in case of the waxy bacteria (tubercle and leprosy) 
and the capsules Of other bacteria may serve a similar purpose 


212 GENERAL BIOLOGY OF MICRO-ORGANISMS 


(pneumococcus). ‘There is some indication that micro-organisms 
may produce special chemical substances to neutralize the agencies 
which the host employs against them. These defensive sub-. 
stances have been designated by Bail as aggressins. Ehrlich 
has also found evidence of the acquirement of immunity to 
chemical substances by certain pathogenic microbes, especially 
trypanosomes and spirochetes, and he ascribes this property of 
the parasites to an alteration of their cell-chemistry. 


4 


‘ 


CHAPTER XII 
REACTION OF THE HOST TO INFECTION 


Facts and Theories.—The host reacts to the presence of a 
pathogenic agent by a number of alterations in its physiological 
activities. Some of these alterations are gross and well known 
as the clinical manifestations of an infectious disease; others 
require special search for their detection; while some, doubtless 
a considerable number, still pass unobserved. Many of these 
changes are susceptible of very accurate‘ observation, and in 
most instances the observed facts are well established. A clear 
understanding of the relation of the various facts to each other 
involves some imaginative reasoning, and various hypotheses 
have been advanced to explain the phenomena observed, and to 
fill in the gaps in our knowledge. The student may need to be 
on his guard not to confuse facts susceptible of observation with 
hypothetical deductions based upon such observations. Both 
have their peculiar value. An understanding of the phenomena 
of pathological physiology must be based upon correct ideas of 
normal physiology and accurate knowledge has not fully replaced 
hypothesis in this latter field. 

Physiological Hyperplasia—Under normal conditions each 
cell of the human body is in close association with other cells 
and with the body fluids, and is subject to the physical and chemi- 
cal stimulation of cells and fluids. One of the effects is apparently 
to restrain the proliferative activity of the cells. When certain 
of these cells are destroyed, or even certain parts of them, this 
restraint is removed, and the natural tendency to proliferation 
asserts itself, resulting in the production of new cells or of new 
parts to replace the old, and usually more than compensates for. 
the loss. This somewhat hypothetical conception, due to Carl 


213 


214 GENERAL BIOLOGY OF MICRO-ORGANISMS 


Weigert, serves to explain tissue hyperplasia and repair following 
exercise or local destruction of tissue. Examples of these phe- 
nomena will occur to the reader. 

Phagocytosis and Encapsulation.—The mere physical mass of 
a parasite within the tissue acts as a foreign body and it becomes 
surrounded by tissue elements. If it is minute, certain cells of 
the body (phagocytes) flow around and ingest it, as was first 
observed by Metchinkoff. If it is larger, the connective tissue 
cells proliferate and surround it, and eventually contract into a 
firm capsule. Further, the tissues produce enzymes capable 
of dissolving many foreign substances introduced in this way 
(parenteral digestion). If the foreign body is insoluble, it will 
remain encapsulated, or, if sufficiently minute, it may be trans- 
ported considerable distances inside wandering cells and eventu- 
ally be deposited in a lymph gland. The wholly passive parasite 
or the dead body of a micro-organism is therefore either digested 
and dissolved, ingested by cells, or encapsulated in fibrous tissue. 
Most infectious agents are not passive in this way, as we have seen, 
but tend actively to grow and multiply, to absorb and utilize food 
material, and, most important of all, to produce various substances 
which stimulate or poison the cells of the host. Against these 
the physical measures of ingestion (phagoctyosis) and encapsula- 
tion are often inadequate defenses and may be entirely useless. 

Chemical Constitution of the Cell—Ehrlich has compared 
the living body cell to a complex chemical molecule; in fact it 
may be said that he regards the living cell as an enormous mole- 
cule, a chemical unit of great complexity. Certain atom groups 
within this. molecule are pictured as relatively very stable and 
they constitute the chemical nucleus (not to be confused with the 
anatomic nucleus). Grouped about this chemically stable center 
are very many, more labile atom groups which readily enter into 
chemical reaction with substances in the surrounding medium. 
The conception is derived directly from well-known facts in organic 
chemistry. For example when benzoic acid, CsH;-COOH, reacts 
with other chemicals the reaction takes place at the reactive group, 


REACTION OF THE HOST TO INFECTION 215 


or side-chain, rather than in the nucleus. The graphic formula 
may illustrate this point better. The six carbon atoms in the ring 


H 
HC 0 
AS a | 
C C—C—OH 
H—C C 
"a 
C oH 
H 


are stable, and a strong chemical reagent, such as phosphorus pen- 
tachloride, reacts with the side-chain without attacking the ring. 
So in the living cell, Ehrich assumes, as a working hypothesis, the 
existence of a wonderfully complex but comparatively stable 
chemical nucleus, with abundant and various more reactive side- 
chains. These latter serve to combine with food materials in the 
surrounding lymph, and these are then ultilized in the cell by an 
intramolecular rearrangement of atoms which is always in prog- 
ress. Useless atomic groups formed in the metabolism of the 
cell are detached and passed off as excretions. These reactions of 
intra-molecular rearrangement and molecular disintegration also 
find their analogues in carbocyclic chemistry. 

Antitoxins.—Von Behring and Kitasato (1890-91) showed 
that animals injected with small non-fatal doses of toxin of the 
tetanus bacillus, produce as a result of this treatment: a some- 
thing which circulates in solution in the ‘blood plasma, which 
is capable of neutralizing the poisonous properties of the tetanus 
toxin. Soon afterward von Behring obtained analogous results 
with the toxin of diphtheria. The protective substances in the 
blood were called antitoxins. The exact chemical composition 
of these substances is unknown. They accompany the pseudo- 
globulin fraction of the plasma in its chemical analysis,’ but the 


1 Banzhaf, Johns Hopkins Hospital Bull., 1911, Vol. XXII, pp. 106-109 


216 GENERAL BIOLOGY OF MICRO-ORGANISMS 
) 


union here is probably a mere physical adsorption or very un- 
stable chemical combination. Ehrlich: explains the formation 
of antitoxin on the basis of his side-chain theory as follows. 
The molecule of toxin attacks the body cell at one of its side-chains 
or receptors which is best adapted to this reaction. In the re- 
sulting intra-molecular_re- 
arrangement the toxin reveals 
itself as a disturbing element, 
causing destruction of that 
portion of the cell to which 
it has become attached. In 
recovering from this disturb- 
ance the cell overcompen- 
sates. by forming an excessive 
number of the particular kind 
of side-chain destroyed, and 
some of the excess side- 
chains are detached, and cir- 
culate in the blood, ready to 
coi hie yah ey raat react with toxin entirely apart 
can Medical Association, 1905, p.955.) a, from the cell which has pro- 
Cell ecepior; toxin molecule; tapi: duced them. ‘These consti 
the toxin molecule; e, haptophore of thecell tute Ehrlich’s receptors of 
acu the first order and their sole 
effect upon the toxin is that of combining with it. The 
free receptors circulating in the blood give it its antitoxic 
property. 
Precipitins.—Other chemical products of bacterial growth 
are attacked and rendered insoluble by products of the body 
cells. Kraus! (1897) showed that animals injected with cultures 
of bacteria produce a substance, or substances, which circulates 
in the blood and is capable of causing a precipitate when mixed 
with the clear filtrate of the cultures of the same bacteria. The 
parenteral introduction of any foreign protein in solution stimu- 
1 Wiener klin. Wochenschr., 1897, X, p. 736. . 


REACTION OF THE HOST TO INFECTION 217- 


lates the production of a substance which will precipitate it. 
These substances, which are called precipitins, resemble enzymes 
in many respects. Thus, the precipitin produced by the injec- 
tion of a milk, causes a change in the milk very similar to that 
caused by rennet. Rennet, however, coagulates milk from various 
animals while the milk pre- 
cipitin is specific, within cer- 
tain limits, for the one kind 
of milk. Precipitation re- 
sults only when the blood 
serum (precipitin) is com- 
bined with the proper 
amount of the culture fil- - 
trate or other protein solu- 
tion (precipitinogen)—when 
too large an excess of one or 
the other is used no precipi- 
tate occurs. Ehrlich ex- 
plains the formation of 


precipitins on the basis of Fic. 88.—Receptors of the second order 


his side-chain theory in the 224 some substance uniting with one of 
y ; them. (Journal of the American Medical 

same way as the production Association, 1905, p. 1113.) ¢, Cell receptor 
: : : of the second order; d, toxophore or zymo- 

of antitoxins was explained. phore group of the receptor; e, haptophore 
The foreign protein stimu- of the receptor; f, food substance or product 


lates the body cells to pro- faptophore of the eal rexepter 
duce specific receptors 

capable of combining with it. In this instance, however, the re- 
ceptor not only combines with the foreign material, but also brings 
abcut a definite change in it which is evidenced by the phenomenon 
of precipitation. The side-chain therefore contains at least two 
distinct atomic groups, one of which serves to combine with the 


precipitinogen, and is specific in nature, and another which brings 


1 Specific precipitin tests have been employed to some extent in determining 
the source of blood stains and of meats. See Citron, Immunity, translated by 
Garbat, Phila., 1914, p. 125. 7 


1 


218 GENERAL BIOLOGY OF MICRO-ORGANISMS 


about the change evidenced by formation of the precipitate. 
The former of these chemical groups is called the combining or 
haptophorous group or haptophore, and the latter is called the 
ferment-bearing or zymophorous group or zymophore. This 
type of side-chain is Ehrlich’s receptor of the second order. It 
is represented in the figure as possessing one smooth branch 
which serves for simple attachment, the haptophore, and one 
branch equipped with saw-teeth to suggest its property of pro- 
ducing chemical change, the zymophore. The precipitin present 
in the blood plasma is supposed to consist of such receptors which 
have become detached from the cell producing them. 

Agglutinins—Gruber and Durham (1896) found that the 
blood of animals suffering from certain infections has the power 
of causing the bacteria involved to clump together and lose their 
motility when it is added to a broth culture or a suspension of the 
bacteria in salt solution. The phenomenon has been observed 
in connection with many bacteria, not only motile but also non- 
motile species, but the most important examples are the typhoid, 
paratyphoid, cholera and dysentery organisms. In typhoid and 
paratyphoid fever the agglutination test is used as an aid in diag- 
nosis of the disease by testing patient’s serum against known 
cultures, and the test with known serum is important in the iden- 
tification of cultures of any of these bacteria. Agglutinins are 
comparatively stable substances although they decompose 
rapidly at 70° to 75° C. When dried they keep for a long time. 
In Ehrlich’s theory, the agglutinins are classed as receptors of 
the second order, along with the precipitins. 

The Phenomenon of Agglutination—Clear fluid blood serum 
to be tested for specific agglutinins is diluted with broth or with 
salt solution to make mixtures containing one part of the serum 
in 5, 10, 20, 40, 80 and 160 parts of the mixture. This is con- 
veniently done by means of the Wright capillary pipette, or 
graduated pipettes may be employed. To each dilution of serum 
an equal amount of a very young (preferably two to six hours 
old) broth culture, or a suspension of an active young agar cul- 


REACTION OF THE HOST TO INFECTION 219 


ture in broth or salt solution, is added. The reaction may be 
observed by mixing small quantities (loopfuls) on a large cover- 
glass and studying the mixture microscopically as a hanging 
drop, or by mixing larger quantities in small tubes and incubating 
them at 37° C. Control specimens free from serum and contain- 
ing normal serum should be set up at the same time for compari- 
son, as many bacteria may be agglutinated somewhat by normal 
serum in a dilution of one to ten, and sometimes the organisms 
in the culture, especially if it is too old, may be already grouped 
together somewhat or may spontaneously clump during the ex- 
periment. Some practice is necessary before one can estimate 
agglutinins reliably and, on the whole, accuracy is more easily 
attained with the macroscopic test. For agglutination tests 
requiring only moderate accuracy, dried blood may be used, the 
dilutions being prepared by comparison of colors with an empirical 
standard. . 

Bactericidal Substances, Alexin.—Nuttall (1886) showed 
that normal blood is capable of killing bacteria and that this 
germicidal property is destroyed by heating the blood to 55° C.. 
for thirty minutes. Buchner confirmed these observations and 
showed further that the germicidal property is resident in the 
serum and not exclusively in the cells of the blood as taught by 
Metchnikoff. To this germicidal substance Buchner gave the 
name alexin, and he ascribed the normal resistance to infection 
exhibited by the healthy animal, as well as the heightened resist- 
-ance of the immunized animal, to this substance. It will. have 
been noted that, historically, these discoveries followed Metch- 
nikoff’s first observations on the phagocytes, and preceded 
the discovery of antitoxins, agglutinins and precipitins, and 
thus presented the first proof of the existence of soluble anti- 
infectious agents. These bactericidal substances are now con- 
sidered to be identical with the bacteriolysins and will be con- 
sidered with them under the more general heading of cytolysins. 

Cytolysins.— Pfeiffer (1896) found that guinea-pigs, when 
injected repeatedly with non-fatal doses of cholera germs, reacted: 


220 GENERAL BIOLOGY OF MICRO-ORGANISMS 


to this treatment by producing a something which would dissolve 
these bacteria. This new property was present in the blood and 
also in the peritoneal fluid. The substance was called bacterioly- 
sin. Subsequent investigators have shown that bacteriolysins 
can be produced for a great variety of micro-organisms, although 
in none can the reaction be better demonstrated than in the 
cholera vibrio originally employed by Pfeiffer. Lysins, or dis- 
solving substances, have been produced for very many other 
kinds of cells also, of which those for red blood cells. (hemolysins) 
are perhaps the most important. It seems to be possible to 
produce a lysin (cytolysin) for any kind of cells by injecting these 
cells into an appropriate animal. , : 

Cytolysins, including bacteriolysins, are active only when 
comparatively fresh. Upon standing for-a day at room tem- 
perature, or upon heating to 56° C. for 30 minutes, the cytolytic 
power disappears. This power is, however, restored in a re- 
markable manner if the cytolysin and the cells to be dissolved are 
injected together into a normal animal, for example into the 
peritoneal cavity of a guinea-pig, or if a fresh normal blood serum 
be added to the mixture in the test-tube. The experiment results 
as follows: 


Immune serum + cholera germs = Bacteriolysis. 
Immune serum (old or heated) + cholera germs = No bacteriolysis. 
Normal serum . + cholera germs = No bacteriolysis. 
Immune serum (old or heated) + normal serum + cholera germs 

= Bacteriolysis. 


This experiment proves that the cytolytic property of the serum 
depends upon the presence of at least two recognizably different 
substances, one of which is present in fresh normal serum and 
in fresh immune serum but is destroyed on standing or by heating, 
and a second which is present in the immune serum and which 
is not destroyed so readily. 

Ehrlich explains the formation of cytolysins by the same 
kind of reasoning as was applied to antitoxins and precipitins. 
The resulting side-chain would be considered of the same sort 


‘ 


REACTION OF THE HOST TO INFECTION 221 


as in the latter class of substances, that is a receptor of the second 
order with a haptophorous group by which to combine with the 
foreign cell, and a zymophorous group to bring about its solution, 
were it not for the observed facts given in the experiment outlined 
above, which demonstrate the presence of two distinct substances 
in the cytolytic complex. A new picture is here necessary and 
it is furnished by making a joint in the arm of the receptor of the 
second order in which the fermentative property is supposed to 
reside, separating off the zymophorous group as a separate sub- 


Fic. 89.—Receptors of the third\order. (Journ. A. M. A., 1905, p. 1369.) ¢, 
Cell receptor of the third order—an amboceptor; e, one of the haptophores of the 
amboceptor with which the foreign body, f, (antigen) may unite; g, the other hapto- 
phore of the amboceptor with which complement, k, may unite; #, haptophore of 
the complement; z, zymophore of the complement. 


stance and leaving a branched figure with two combining or 
haptophorous elements, one capable of combining with the foreign 
cell and the other capable of combining with the cytolytic ferment 
of normal serum and so bringing its action to bear upon that 
particular cell. The receptor of the third order is called, in 
accordance with this conception of its relationships, amboceptor, 
because it acts as a receptor at two points. It is also called 
intermediary body, immune body and sensitizer. The other com- 
ponent of the lytic complex, which is thermolabile and which is 


222 GENERAL BIOLOGY OF MICRO-ORGANISMS 


present in normal serum is called complement or cytase and 
by some authors (Bordet) alexin.! It will be noted that only a 
part of the cytolysin is produced by the body in its reaction to 
invasion, namely, the immune body. 

Deviation of Complement.—Neisser and Wechsberg observed 
that the bactericidal power of a given immune serum (bacteriolytic 
amboceptor), when combined with a constant amount.of normal 
serum (complement) and a constant amount of a bacterial sus- 
pension (antigen), increased progressively with progressive dilution 
of the immune serum to a certain point, after which it diminished 
again. The following data taken from Citron illustrate’ the 
experiment: 


Fresh serum Colonies produced 
Teale eltee Gallia | Stood | otic | Saaee 

OG CC) 1/5000: o cacwend nan 1/100 ¢.c. 0.5 C.C. Many thousand 
O15 GC) T/§SO0G ns scdiona a scene 1/5000 c.c. 0.5 C.C. Many thousand 
0.5 C.C. 1/§000..........% ..| 1/20000 ¢.c. 0.5 C.c. 200 
0.5 C.C. 1/5000.............| 1/30000 ¢.c. 0.5 C.C. ° 
OMG Gils T/SOOOs ore eee ys be 1/50000 ¢.c. 0.5 C.C. 60 
0.5 C.c. 1/§000.............] 1/200000 €.c. 0.5 C.C. Many thousand 


Neisser and Wechsberg have undertaken to explain this 
result by supposing that the excessive number of amboceptors 
present in the more concentrated solutions of immune serum 
hinders cytolysis because some of them combine with the antigen 
by means of their cytophile groups while others are combining 
’ with the complement by means of their complementophile groups, ° 
and as a result the mixture contains combinations of amboceptor 
with antigen, and of amboceptor with complement, but practically 
no combinations of the three elements together. There are 
grave reasons for questioning the accuracy of this assumption, 


1 This use of the term alexin would seem to-be undesirable, for Buchner employed 
the term to designate the whole bactericidal or cytolytic complex before the possi- 
bility of recognizing two separate elements was clearly recognized. 


REACTION OF THE HOST TO INFECTION 223 


as it has been shown by Bordet that: amboceptor does not unite 
with complement in the absence of antigen. It seems more 
probable that some other factor, such perhaps as a marked agglu- 
tination of the bacteria in the stronger solutions, may serve to pro- 
tect them from the bacteriolytic action. 

Fixation of Complement.—As has been mentioned, it is pos- 
sible to produce cytolysins for red blood cells. This is commonly , 
done by injecting the washed blood corpuscles of a sheep (o.1 c.c. 
+ 0.5 c.c. salt solution) into a rabbit intravenously three or four 
times at intervals of five days. The serum of the rabbit becomes 


ee XK is 


Fic. 90.—Illustrating the conception of deviation of complement. a, Amboceptor; 
6, antigen; k, complement. 


strongly hemolytic for sheep’s cells. The blood is drawn from 
the carotid artery, the serum separated, rendered perfectly clear 
and after heating to 56° C. for 30 minutes is stored in hermetically 
sealed ampoules containing 1 c.c. each, in a low temperature 
refrigerator. When this hemolytic amboceptor is diluted to the 
proper point, which must be ascertained by trial and error, it 
will just cause the complete hemolysis of a definite quantity of 
washed sheep’s corpuscles (0.2 c.c. of a 5 per cent suspension) 
when combined with 0.1 c.c. of a ro per cent solution of fresh 
normal serum of guinea-pig (complement). The mixture of this 
quantity of the immune serum, which may now be called one unit 
of hemolytic amboceptor, with 0.2 c.c. of freshly prepared 5 per 
cent suspension of washed sheep’s corpuscles produces a reagent 
which serves for the detection of complement and the approxi- 


224 GENERAL BIOLOGY OF MICRO-ORGANISMS 


mate estimation of its amount in an unknown mixture. By 
the use of such a reagent it is possible to show that complement 
is destroyed or used up in various specific cytolytic, proteolytic, 
and precipitin reactions. Thus Bordet and Gengou mixed to- 
gether typhoid bacilli (antigen), heated typhoid-immune serum 
‘(amboceptor) and fresh normal serum (complement) and incu- 
bated the mixture. After an hour the hemolytic.amboceptor 
and sheep’s blood cells were added and incubation continued. 
No hemolysis resulted, showing that the complement added in 
the first place had been used up, “fixed,” as a result of a reaction 
with the typhoid bacilli and typhoid amboceptor. This is the 
phenomenon of fixation of complement. Obviously it lends itself 
to use as a test for the presence of a:specific antigen or for the pres-_ 
ence of specific amboceptor. Its more definite application will 
require subsequent mention. 
Opsonins.—Wright and Douglas (1903) showed that blood 
serum contains a something which affects bacterial cells, soaked 
in the serum, in such a way that they are more readily ingested 
by the living leukocytes. To this substance they gave the 
name ‘‘opsonin” (opsono, I prepare victuals for), Substances 
of this sort are present in normal blood, but are increased as a 
reaction following infection. It would seem that more than 
one substance may act upon bacterial cells in this manner, for 
Neufeld has shown that the opsonic power of normal serum may 
be destroyed by heating to 56° C., whilé the similar property of 
immune serum remains after this treatment. It is not yet con- 
clusively proven that opsonins are separate substances entirely 
distinct from bacteriolysins and agglutinins, but it has been shown 
that opsonic power of a serum does not correspond in its con- 
centration to that of the other antibodies, and some other element 
must, therefore, be a factor. Hektoen considers the opsonins 
to be distinct bodies, different from lysins and agglutinins. 
The study of opsonins has done much to bring about harmony 
between the followers of Metchnikoff, with their tendency to 
emphasize the importance of phagocytosis, and the followers 


‘REACTION OF THE HOST TO INFECTION 225 


of Buchner and Ehrlich, who fixed their attention largely upon. 
the substances dissolved in the body fluids. 

Anti-aggressins, Specific Proteolysins.—Various substances 
produced in the body as a result of infection show particular 
ability to combat the effects of the soluble products of the para- 
site to which the name aggressins has been given (see page 212). 
Knowledge of these substances and. their behavior is still some- 
what incomplete, but they seem to be particularly concerned 
with the parenteral digestion of foreign proteins, a process in which 
cytolysis may be regarded as a beginning stage. Whereas, 
however, cytolysis is concerned with the disintegration of formed 
material, these substances now under consideration act particu- 
larly upon proteins already in solution. In many instances the 
products of the first stages in this parenteral digestion are toxic 
(disintegration of tuberculin and of egg-white), and some of the 
symptoms of infectious disease, such as fever, have been ascribed 
to them. In their general characters these lytic substances are 
wholly analogous to the cytolysins and their action is due to at 
least two components, an amboceptor and a complement. 

Source and Distribution of Antibodies——The exact source 
of the antibodies dissolved in the body fluids is unknown. All 
agree that they are derived from cells. Metchnikoff regards 
the phagocytic cells as the important source; Ehrlich does not 
specify, but it would seem, in accordance with his theory, that 
any cell capable of being affected by the foreign substance should 
be capable of throwing off cell receptors (antibodies) to combine 
with it. Many investigators consider antibody formation to be a 
common property of many kinds of cells, but more especially of rela- 
tively undifferentiated cells such as those of the connective tissue. : 

Antibodies are present in greatest concentration in the blood 
and lymph. They are absent or present only in small amount 
in the serous fluids of the pleural, pericardial, peritoneal and 
joint cavities, and in the cerebrospinal fluid. Parasites in 


1 See Flexner, Harbin Lectures, Journ. of the State Medicine, March, April, May 


Igr2. 
15 


226 GENERAL BIOLOGY OF MICRO-ORGANISMS 


these locations are less readily influenced by antibodies circulating . 
in the blood, so that localized infections may continue in these 
regions in spite of a considerable concentration of antibodies in 
the body generally. 

Allergy.—Allergy is a term snvented by Von Pirquet to 
designate the condition of altered reactivity on the part of the 
body which comes about as a result of infection. A few of the 
phenomena which may be included under this term have been 
considered above in this chapter. Many of these alterations in 
bodily function are manifestly of advantage to the host ih limiting 
the activities of the parasite, neutralizing its poisonous products, 
and even in destroying and removing the parasite itself. Some of 
them, such as specific precipitation, seem to serve no important 
purpose, while others, such as cytolysis and proteolysis, actually 
lead sometimes to results very harmful to the host, although 
their usual effect is favorable. Many of the recognized weapons 
which the body employs in its battle against parasites are still 
imperfectly understood, and there are doubtless many factors 
involved in this relation which are not yet capable of definite 
recognition. Of those agents mentioned above, the phagocytes 
are ready for immediate defense as soon as the body is invaded 
by the parasite. Hyperplasia and encapsulation require more 
time, probably one to four weeks. The chemical antibodies, » 
antitoxins, agglutinins, cytolysins and opsonins, although possibly 
present in small amounts in the normal body fluids, become 
definitely increased in from eight to twelve days after the entrance 
of the parasite, an interval approximately equal to the incubation 
period of some infectious diseases. These various agents have 
much to do in determining the manifestations and course of the 
disease as well as the final outcome, and as we shall see, they also 
play a part in immunity.’ 


CHAPTER XIII 


IMMUNITY AND HYPERSUSCEPTIBILITY. THEORIES 
OF IMMUNITY 


Immunity.—Immunity is that condition of a living organism 
which enables it to escape without contracting a disease when 
fully exposed to conditions which normally give rise to that disease. 
Immunity may depend upon many different factors, or upon 
only one of a great variety. In general, we shall find that it 
depends very largely upon those factors which we have already 
considered in the preceding chapters, such as the possession of 
anatomical structures or habits of life which render invasion by. 
the particular parasite impossible, or the possession of a body 
structure, physically or chemically not adapted for the growth 
of the particular disease virus, or the ability to harbor the par- 
ticular parasite as a commensal without suffering injury, or the 
ability to react against the invading parasite and destroy it by 
phagocytosis or by cytolysis, neutralize its poisons by antitoxins, 
or limit its activity by encapsulation. Immunity is ordinarily 
considered under two heads, Natural Immunity, or that present 
as a part of the individual’s birthright, and Acquired Immunity, 
that which follows as the result of some experience of theindividual. 

Immunity of Species.—Natural immunity to certain diseases 
is possessed by certain species of animals. Where the morphology 
and physiology is quite different from that of the usual victims 
of the disease, immunity might be expected. Thus cold-blooded 
vertebrates, fish, amphibians and reptiles, are immune to many 
diseases of mammals, apparently because of the different tem- 
perature of their tissues. In other instances the difference 


in resistance between two species of animals seems to be correlated 
227 


228 ' GENERAL BIOLOGY OF MICRO-ORGANISMS 


with difference in food habits. Thus the carnivorous mammals 
are relatively insusceptible to anthrax and tuberculosis, diseases 
natural to the herbivora. Many infectious diseases of man 
are not readily transmissible to animals, for example, typhoid 
fever, syphilis, pneumonia, and in some instances it has so far 
been impossible to infect animals, as for example with scarlet 
fever and gonorrhea.* 

Racial Immunity.—Within a species there is moreover a 
racial difference in resistance to natural infection. Thus the 
pure-bred dairy cattle are more susceptible to tuberculosis than 
other cattle,-and Yorkshire swine are relatively. less susceptible 
to swine erysipelas. In man, the relation of race to susceptibility 
is not very clear. The examples of supposed racial immunity 
have not proved to be so definite as has been assumed at first. 
Thus the supposed immunity of African natives to syphilis has 
vanished with their increasing contact with civilization and 
with this accompanying disease. In the case of malaria the 
supposed racial immunity of negroes seems to be an acquired 
immunity due to severe attacks of the disease in childhood. 
There is, however, some evidence that. prolonged contact with a 
disease through many generations may result in a relative resist- 
ance, so that the disease assumes a milder form in such a race of 
people—a sort of inherited acquired immunity. Such considera- 
tions have been brought forward to explain the relatively high 
resistance to tuberculosis shown by the Hebrews as compared 
with the American Indians. \ 

Individual Variations.—Individual variations in resistance 
to infection are commonly observed. They may depend in part 
upon age, condition of nutrition, fatigue, exposure or intoxica- 
tion, but they are ascribed also to differences in anatomical 
structure (shape of the thorax in tuberculosis). Individuals 
especially susceptible to a disease are said to possess an idiosyn- 
crasy for it. The physiological mechanisms upon which varia- 
tions in individual resistance depend are not clearly understood. 

1 Kolle und Wassermann, II Aujflage, Bd. IV, p. 693 (1912). 


IMMUNITY AND HYPERSUSCEPTIBILITY 229 


¥ 


Doubtless, the number and activity of the white blood cells and 
the nature and amount of bactericidal substances in the blood 
play a part in some instances. 

Acquired Immunity.—Acquired immunity results from some 
experience affecting the individual, either an infection which the 
individual has survived or‘some artificial procedure of immuniza- 
tion. There are recognized two different kinds of acquired 
immunity, first, active immunity which is due to the activity 
of the cells of the individual immunized, and second, passive 
immunity which is produced by introducing into the body, 
material (blood serum) from another animal, which contains 
substances conferring at once an immunity upon the new 
individual. 

Active Immunity.—Active immunity may be acquired by an 
attack of the disease. This immunity may endure for a lifetime 
in some instances (yellow fever, small-pox, scarlet fever) or for 
many years (typhoid fever) or it may be very evanescent (ery- 
sipelas, pneumonia, influenza). Some diseasés were at one 
time so universal that few escaped them, and individuals used 
to be purposely exposed or inoculated in order to contract the 
disease and gain the resulting immunity. Inoculation of small- 
pox seems to have been practised in China about 1000 A. D. and 
in India as early as the twelfth century, and it was introduced into 
Europe in 1721 by Lady Montague and was employed very 
extensively in Europe and America during that century. 

Active immunity may also be produced without causing a 
definite attack of the disease. This may be accomplished in a 
variety of ways. Fully virulent micro-organisms may be intro- 
duced into a part of the body unfavorable to their development. 
The subcutaneous injection of cholera cultures according to the 
method of Ferran and Haffkine has proven to be practically 
without danger, and results in immunity. The same principle 
is ultilized in immunizing cattle against pleuro-pneumonia.' 
Introduction of virulent organisms in very minute doses has been 

1 Kolle und Wassermann, IT Auflage, Bd. I, S. 928 (1912). * 


230 GENERAL BIOLOGY OF MICRO-ORGANISMS | 


employed to immunize against rabies (Hodgyes method), and 
against tuberculosis by Webb. In most diseases these methods 
are regarded as too dangerous for extensive use. 

. Living virus, altered in its virulence, was first used by Edward 
Jenner, when he inoculated with cow-pox (vaccinia) and induced 
immunity to small-pox. Cow-pox is doubtless due to the organism 

which causes small-pox, attenuated by its life in the body of the 
cow. Viruses artificially cultivable are attenuated by a variety 
of procedures, and are employed to induce immunity. ‘Pasteur’s 
vaccine for anthrax, for chicken cholera and possibly the treatment 
of rabies with dried spinal cord, are examples of the application of 
this principle. Virus of extraordinary virulence is sometimes in- 
jected after previous treatment with attenuated organisms, in 
order to confer a higher degree of immunity. Thus Pasteur 
employed the most virulent rabies virus obtainable, varus fixé, in 
the immunization against rabies. 

Living virus, of full virulence, but apparenty influenced in 
some way by the body fluid containing it, is employed in immuniz- 
ing against rinderpest and against Texas fever. The bile of an 
animal dying of rinderpest is injected subcutaneously in doses of 
ro c.c. into cattle. Kolle has shown that the virus can be sepa- 
rated from such bile in fully virulent condition; so it appears 
that scme constituents of the bile restrain the activity of the 
virus. In Texas fever, blood of young animals containing rela- 
tively few of the parisites is used to inject new animals. 

Immunization by injection of dead microbic substance is now . 
extensively employed in the prophylaxis of cholera, typhoid fever 
and plague. As a result of such injections there is a marked in- 
crease in specific agglutinins and bacteriolysins in the blood. The 
principle of general immunization is also employed with some suc- 
cess in the treatment of subacute, chronic or recurrent local 
infections, the production of antibodies and their circulation in 
the blood and lymph exerting a favorable effect upon the local 
lesions. The emulsions of dead bacteria employed are called 
bacterial vaccines. 


IMMUNITY AND HYPERSUSCEPTIBILITY 231 


The soluble products of bacterial growth are injected into 
animals to immunize them, especially in the case of diphtheria 
and tetanus, the bacteria of which produce very powerful soluble 
toxins. Asa result of this treatment antitoxins are produced and 
circulate in the blood of the animal. 

Bacterial extracts, either those contained in inflammatory 
exudates, the so-called aggressins of Bail, or extracts obtained by 
soaking bacteria in blood serum or in distilled water, the so-called 
artificial aggressins of Wassermann and Citron, have proved of 
value in experimental immunization of animals against many dif- 
ferent bacteria. It is claimed that the reactions to injection are 
exceptionally mild, while the resulting immunity is more solid. 
Certain products of the disintegration of typhoid bacilli have been 
obtained by Vaughan, which possess considerable immunizing 
. power, but apparently only slight toxicity. None of these bac- 
terial extracts has yet passed beyond the experimental stage in the 
immunization of man against a disease. 

A certain slight grade of immunity may be secured in some 
instances by procedures which seem to bear no relation to the 
specific micro-organisms in question. Thus the injection of cul- 
tures of B. prodigiosus and B. pyocyaneus results in an increased 
resistance to infection with anthrax. Similar increased resistance 
has been observed to follow a simple surgical procedure, such as 
section of the sciatic nerve. The explanation of these results is 
not clear, but perhaps the effect may be attributed to a general 
stimulation of the body defenses, especially the phagocytes. 

Passive Immunity.—Passive immunity. is produced by inject- 
ing into the body a fluid taken from another animal, which con- 
‘tains antitoxins, bacteriolysins, opsonins or other substances known 
as immune bodies. The animal which furnishes the immune 
bodies must be first actively immunized, and it possesses an ac- 
tive immunity. If its blood plasma be drawn and injected into 
a child, the child acquires a borrowed immunity without the 
necessity of any active participation of its own cells in the pro- - 
duction of immune bodies. The possibility of producing such 


232 GENERAL BIOLOGY OF MICRO-ORGANISMS 


passiveimmunity has been demonstrated in anumber of diseases. In 
some instances the effect of the serum is antitoxic (diphtheria and te- 
tanus), in others it is batteriolytic (cholera), while in other instances 
the nature of the dominant antibodies is not definitely. known. 

Combined Active and Passive Immunity.— Various procedures 
have been devised to produce immunity by introducing at, or 
nearly at, the same time the infectious agent or its products and 
the serum of an immune animal containing protective substances. 
The combination of immune blood with virus of full strength is 
used in immunizing animals against rinderpest, foot-and-mouth 
disease and hog cholera, all being diseases due to filterable agents; 
arid also in immunizing hogs against hog erysipelas (B. rhusio- 
pathie). The combined injection of attenuated virus and immune 
serum is employed especially in Sobernheim’s method of preventive 
inoculation against anthrax. Besredka has employed dead 
bacteria combined with their specific immune serum in immunizing 
against typhoid fever, plague and cholera. 

The Mechanisms of Immunity.—Certain biological factors 
in the phenomenon of immunity are now clearly recognizable 
and readily demonstrable. The activity of the phagocytes, first 
emphasized by Metchnikoff and believed by him to be the sole 
important factor in the defense of the body, is easily observed 
in immunity to many diseases. The dependence of phagocytic 
activity upon dissolved substances in the body fluids (opsonins) 
is also demonstrated beyond doubt. Phagocytosis is, perhaps, 
the factor of most general operation in immunity to all sorts of 
disease. The antitoxins stand forth prominently as powerful 
factors in immunity to two important diseases, diphtheria and 
tetanus, and the bacteriolysins are undoubtedly of greatest im- 
portance in the case of Asiatic cholera, and probably also in ty- 
phoid and plague. In most instances the immunity seems to 
depend upon several different factors, phagocytosis, opsonins, 
bacteriolysins, antitoxins, and perhaps substances of unknown 
nature. In some instances of immunity there is no particular 
excess of these immune bodies demonstrable in the blood, and 


IMMUNITY AND HYPERSUSCEPTIBILITY 233 


nearly always an immunity remains long after such an excess 
has disappeared. It would seem that the ability of the cells of 
the body to respond promptly to invasion is often developed by 
experience with one such invasion, and that this uy) may re- 
main for a long time as a factor in immunity. 

Hypersusceptibility or Anaphylaxis—If a guinea-pig abe in- 
jected with a small amount of a protein, such as egg-albumen or 
blood serum of the horse, and then after an interval of ten to 
twenty days be injected with a second dose of the same protein 
of good size (0.5 to 5 grams), the animal usually develops symp- 
toms of nervousintoxication and often dies within a half hour. In- 
asmuch as normal guinea-pigs withstand enormous doses of such 
protein substances, it is evident that the first injection has brought 
about some change in the animal, an altered reactivity, which 
results in the intoxication after the second dose. That this phe- 
nomenon of hypersusceptibility or anaphylaxis (=against pro- 
tection) bears a definite relation to immunity may be illustrated 
by an experiment in which typhoid bacilli are substituted for 
the soluble protein. If a guinea-pig be immunized by repeated 
doses of the killed micro-organisms he is able to resist inoculation 
with an ordinarily fatal dose of the living germs, which are quickly 
killed and dissolved by the specific bacteriolysins in the body 
fluids. However, if such an immune guinea-pig be injected with 
a proper dose of dead organisms, which would not kill a normal 
animal, he may quickly succumb. The ability of the body fluids 
of the immune animal to disintegrate the bacterial cells rapidly 
would seem to be the factor upon which depends not onlv its 
immunity to the small dose of living germs, but also its exagger- 
ated 'sensitiveness to dead germ substance. The products of the 
rapid parenteral digestion of the foreign protein would seem to be 
the cause of the symptoms of intoxication. The essential unity 
of the substances upon which immunity and anaphylaxis depend 
has been emphasized by Von Pirquet! and his co-workers.’ 


1 Von Pirquet: Allergy. Archives of Internal Medicine, 1911, Vol. VII, pp. 259-288; 
Pp. 383-436. 


234 GENERAL BIOLOGY OF MICRO-ORGANISMS 


Theories of Immunity.—Early theories of immunity were 
based upon meager observations. The idea that an attack of a 
disease left behind in the body something which prevented the 
subsequent entrance of that disease was formulated by Chauveau 
in 1880 as the so-called retention hypothesis. In the same year 
Pasteur expressed the idea that an attack of a disease removed 
something from the body and so exhausted the soil as far as that 
particular disease was concerned. Neither of these ideas was 
new at that time, and neither of them pretended to any very 
definite or specific application to phenomena observed in immu- 
ity, but only to the general phenomenon of immunity itself. 
The discovery of phagocytosis by Metchnikoff in 1884 was the 
first observation of a definite phenomenon which appeared to 
explain the facts of immunity. The phagocytic theory, which 
grew out of this observation, was an attempt to ascribe immunity 
in general to this one phenomenon of phagocytosis. With the 
observation of the bactericidal substances in solution in the blood 
plasma by Nuttall and by Buchner, of the antitoxins by von 
Behring and the bacteriolysins by Pfieffer, there developed at- 
tempts to ascribe all the observed facts of immunity to these 
factors, resulting in the alexin theory and the antitoxin theory 
of immunity. More intimate study of the dissolved immune 
bodies lead to the formulation of a hypothesis to explain their 
formation, composition and action, the side-chain theory of Ehr- 
lich, which has been of great value as a working hypothesis and 
as a central conception about which to arrange the observed facts 
relating to these dissolved substances. The elementary concepts 
of this theory have been given in the preceding chapter. 

In brief, Ehrlich pictures the living cell as a chemical unit 
‘possessing numerous and varied combining groups or side-chains 
capable of uniting with substances in contact with the cell. The 
toxin molecule is conceived as a substance containing at least 
two distinct chemical groups, one which serves for attachment 
to the side-chain of the cell and the other serving to bear the poison- 
ous properties. The union of the toxin with the cell results in 


IMMUNITY AND HYPERSUSCEPTIBILITY 235 


destruction of the side-chains attacked, and in regenerating these 
the cell over-compensates, the excess side-chains, receptors -of 
the first order (see page 216), being set free into the blood and con- 
stituting the antitoxin, which is capable of neutralizing toxin there 
or in the test-tube. The assumption of two chemical groups in 
the toxin molecule is strenghtened by the observation that diph- 
theria toxin changes on standing so that its poisonous property is 
much diminished without corresponding loss of ability to combine 
with antitoxin. Such changed toxin, in which the haptophorous 
group persists while the toxophorous group has degenerated, is 
called toxoid. In order to explain the formation and structure of 
agglutinins and precipitins, Ehrlich assigned a more complex com- 
position to the side-chains which constitute these substances, lead- 
ing to the conception of a receptor of the second order (see page 
217), withits haptophorous and zymophorous groups. In the case 
of the cytolysins, a further amplification of the idea was necessary 
to explain the observed fact that the cytolysis is due to two com- 
ponents, one of which is a thermolabile, normal constituent of the 
blood and not increased as a result of immunization, the other be- 
ing a thermostable substance which is produced as a result of the 
immunization process. This latter immune body, the receptor of 
- the third order, was therefore pictured as a double receptor (ambo- 
ceptor) capable of attaching on the one hand the foreign body 
(antigen) and on the other the normal component necessary to 
complete the lytic complex, to which component the name comple- 
ment was given. 

With the recognition of opsonins by A. E. Wright in 1903, 
the opposing theories of the French and the German schools be- 
gan to be reconciled, and the relatively simple and largely hypo- 
thetical theories of immunity began to give way to a more exact 
and necessarily more complex science of immunology. Bordet 
and his pupils deserve credit for leading the reaction against too 
slavish adherence to theory in the study of immunity. Our 
modern ideas are no longer confined within the scope of any one 
theory and it is necessary to recognize the existence of a great 


236 GENERAL BIOLOGY OF MICRO-ORGANISMS 


variety of phenomena in the interaction of the host cells and their 
secretions on the one hand with the parasites and their chemical 
products on the other. The elementary conceptions of immun- 
ology and the primary language of the science are derived from 
the old theories, especially from Ehrlich’s theory, and these theo- 
ries are an essential part of the introduction to immunology.! 


1 For a concise presentation in English of facts\and practical experiments re- 
lating to immunity, the student is referred to Citron, Immunity, translated by 
Garbat, Philadelphia. 


PART Ill 
SPECIFIC MICRO-ORGANISMS 


CHAPTER XIV 


THE MOLDS AND YEASTS AND DISEASES CAUSED BY 
THEM 


Mucor Mucedo.—This is the most common species of mucor, 
especially about barns and on manure. It produces a network 
of threads (mycelium) in the substratum, and zygospores are pro- 
duced here by the union of two cells. The aérial hyphe are 
vertical, about 10 cm. in length and bear a spherical spore sac 
(sporangium) at the end. The sporangium is at first yellow, 
later brown and finally black and covered with crystals. The 
contained spores are 4 to 6u wide by 7 to 10m long. It is sapro- 
phytic and may be found as a contamination on culture media. 

Mucor Corymbifer.—Lichtheim found this mold growing on 
a bread-infusion gelatinvas an accidental contamination. The 
growth is at first white and later gray. The spore-bearing hyphe 
are long and irregularly branched, and each branch bears a pear- 
shaped sporangium Io to 704 in diameter. The contained spores 
are small (2X3). Intravenous injection of the spores into rab- 
bits causes severe nephritis and death in two or three days. 
The mold has been found growing as a parasite in the auditory 
canal. 

More than a hundred species of Mucor have been described 
and several of them cause disease and death when injected into 


. animals. 


if 237 


238 SPECIFIC MICRO-ORGANISMS 


3 J 4 


Fic. 91.—Mucor mucedo. 1, A sporangium in optical longitudinal section: 
c, columella; m, wall of sporangium; sp, spores. 2, Aruptured sporangium with only 
the columella (c) and a small portion of the wall (m) remaining. 3, Two smaller 
sporangia with only a few spores and no columella. 4, Germinating spore. 5, 
ruptured sporangium of Mucor mucilaginus with deliquescing wall (m) and swollen 
interstitial substance (z); sp, spores. (From Jordan after Brefeld.) 


i 


Fic. 92.—Mucor cor: 


MOLDS AND YEASTS AND’ DISEASES CAUSED BY THEM 239 


Aspergillus Glaucus.—This is very widely distributed in 
nature, occurring on fruits, moist bread and other food substances 
and very frequently as a contamination in laboratory cultures. 
The aérial spore-bearing hypha (conidiophore) is erect, about 
t mm. long, swollen at the end to a diameter of 20 to gou. On 
the surface of this spherical head are numerous closely packed 
spore-bearing sterigme, each of which bears at its tip a chain of 
spherical spores (conidia) which ! 
are budded off from it. The 
conidia are gray to olive green 
in color. Ascospores are also 
produced, grouped together as 
yellow masses, called perithe- 
cia, on the surface of the 
medium. The mold is not 
pathogenic. Probably a con- 
siderable number of different 
species have been included 
under this name. 

Aspergillus Fumigatus— 71°. p2-Asporius fumians trom the 
The growth of this mold is at 
first bluish and later grayish-green. Itis widely distributed. The 
sterigmz are unbranched, thickly set on the swollen end of the 
spore-bearing hypha. The conidia measure 2.5 to 3u. The for- 
mation of ascospores has also been observed. Aspergillus fumi- 
gatus plays a part in the heating of hay and sprouting barley, 
and is the most common of the pathogenic aspergilli. It infects 
doves and other birds naturally, sometimes causing veritable 
epidemics, and the disease has been observed int bird fanciers, 
in whom it runs a clinical course very similar to that of pulmonary 
tuberculosis. Fragments of the mycelium are found in the spu- 
tum. Doubtless the human disease is contracted from the birds 
in these cases. This mold has been found as the apparent cause 
of inflammation in the auditory canal in a large number of cases 
and in the nasal toss in afew instances. Various other mammals 


240 SPECIFIC MICRO-ORGANISMS 


are susceptible to inoculation and natural infection has been ob- 
served in horses, cattle, sheep and dogs. 
Many other species of pathogenic aspergilli have been de- 
scribed, of less frequent occurrence than A. fumigatus. — 
Penicillium crustaceum (glaucum) 
is the commonest contaminating mi- 
cro-organism met with in the labora- 
tory, and is probably the most widely 
distributed mold. Ascospores, similar 
to those of Aspergillus glaucus have 
keen observed, but they are rarely 
produced. The aérial fruiting hypha 
(conidiophore) is erect, septate and 
branched at the upper end like a brush. 
At the end of these branches are bot- 
tle-shaped stergmz from which the 
conidia are constricted off. The 
growth is at first white and then it 
becomes blue-green, the development 
of color being at the center. It is 
not pathogenic, but the extracts from 
cultures of some varieties are poison- 
rene ous when injected into arimals. It 
Fic. 94.—Penicillium crusta- , ‘ ae : 
ceum. Conidiophore with verti- is possible that several distinct species 
a ee Se wn Ste have been included under this one 
conidia. 540. (From Jordan name of Penicillium crustaceum. One 
after Strasburger.) a Fac aerate 
nearly related organism, Penicillium 
rocqueforti, Thom, is an important agent in the ripening of 
Rocquefort cheese. 
Claviceps Purpurea.—This is a fungus parasitic upon rye and 
a few other plants. The spores gain access to the flower of rye 
and develop a mycelial mass which grows in the utricle, dis- 
placing the grain, the rudiment of which lies above the mass of 
the mold. Closely packed conidiophores produce oval conidia 
and at the same time secrete a sweet milky fluid which attracts 


MOLDS AND YEASTS AND DISEASES CAUSED BY THEM 241 


insects and thus furthers the distribution of the parasite. Later 
the mycelial mass produces sclerotia, which are masses of thick- 
walled cells containing starch and oil together with specific poi- 

_ sonous substances, and the whole becomes dry and hard with black 
outer covering, forming the ergot grain, which is considerably 
larger than the normal rye grain. In autumn this falls to the 
ground and remains until spring, when numerous red stalks grow 
out of it. Upon the swollen ends of these stalks, abundant as- 
cospores are produced, and these serve to infect again the flowers 
of the new crop of rye. 

This fungus is of great importance as the source of the drug, 
ergot, and as a cause of food poisoning, ergotism, in certain coun- 
tries. It is one example of a mold parasitic upon higher plants. 
There are very many different species of such parasitic fungi, 
and they are probably the best known microbic agents causing 
diseases of plants.1 

Saccharomyces Cerevisiz.—This organism is the type of the 
true yeasts. The cell is spherical or ovoid and multiplies by 
budding. Ascospores are produced, usually four to eight in a 
single cell. Saccharomyces cerevist@ is found widely distributed, 
especially on fruits and in sugar-containing substances. It has 
been used for centuries in the leavening of bread and in the al- 
coholic fermentation. Varieties of the species are distinguished 
by differences in physiological activity and especially in respect 
to the amounts of alcohol which they produce. 

Material for study may be obtained from commercial com- 
pressed yeast, which contains vegetating cells of Saccharomyces 
mixed with other organisms including as a rule Oidium lactis and 
various bacteria, or from commercial dried yeast in which the 
ascospores are present. Pure cultures may be obtained by plating* 
this material on nutrient gelatin. Saccharomyces is found in the 
gastric juice at times and is evidently capable of multiplying within 
the stomach when the acidity of the gastric juice is diminished. | 

1 For a consideration of molds in relation to plant pathology, see Massee, Diseases 


of cultivated plants and trees, New York, 1910. 
16 , 


242 SPECIFIC MICRO-ORGANISMS 


Coccidioides Immitis.—Posadas! and Wernicke? first ob- - 
served in human lesions the doubly contoured spherical forms of. 
this organism, which multiplies in the tissues by endogenous 
spore formation. They regarded the parasite as a protozoon. 
The organism was named Coccidioides immitis by Rixford and 
Gilchrist in 1896 and it was recognized as a mold by Ophiils 
and Moffitt in 1900. Wolbach, in 1904, made an extensive study 


50 Me 
‘F1G. 95.—Coccidioides immiltis: a, band c represent the doubly contoured spheres 


seen in fresh pus; d represents the same organism as a after incubation for 24 hours 
at 33° C. in a-hanging-block culture. (After MacNeal and Taylor.) 


of the organism in cultures and by inoculation of animals. In 
1914, MacNeal® and Taylor followed the transformation of the 


1Posadas, Infectiose generalisierte Psorospermosis. Buenos Aires, 1897; 
Ref. in Monatshefte f. prakt. Dermatol., 1898, 27, p. 593; Psorospermiose in- 
fectante généralisée. Revue de Chirurgie, 1900, 21, p. 276. 

2 Wernicke, Ueber einen Protozoenbefund bei Mycosis fungoides? Centralbl. 
f. Bakt., 1892, 21, p. 859. 4 

3 MacNeal and Taylor, Coccidioides immitis and coccidioidal granuloma. 
Journal of Med. Rsch., 1914, 30, p. 261. References to previous literature are given 


in this paper. 


MOLDS AND YEASTS AND DISEASES CAUSED BY THEM 243 


doubly contoured spherical parasitic form into mycelial growth in 
the agar hanging block inoculated with pus. The reverse trans- 
formation of the mycelium into spherical forms was also followed 
in the inflammatory exudate of animals inoculated with the 
mycelial growth and finally it was shown that, by the exclusion of 
air, the parasitic spherical form could be made to continue for a 
time its multiplication by endogenous spore formation in artificial 
culture. 

Coccidiodes immitis causes a highly fatal disease of man, 
coccidioidal granuloma, protean in its clinical manifestations, 
usually chronic in course and often presenting in the histology of 
its lesions the most perfect mimicry of tuberculosis. The parasite 
in such lesions is a spherical body 20 to 35 u in diameter, with a 
doubly contoured wall, filled with a granular protoplasm, some- 
times vacuolated, sometimes segmented. Guinea pigs are sus- 
ceptible to inoculation. The disease seems to be confined to the 
western hemisphere and a large proportion of the reported cases 
have developed in California. The mode of transmission of the 
disease and the possible existence of the parasite in the external 
world under natural conditions have not been ascertained. 

Bottytis Bassiana.—This mold was shown to be the cause of 
muscardine, a disease of silkworms, by Bassis and Audouin in 
1837, a discovery following closely the recognition of the itch 
mite, Sarcoptes scabei, as the cause of scabies in 1834. The in- 
fected silkworm becomes sluggish and dies, and the aérial hyphe 
of the fungus grow out from its surface and pinch off round or 
pear-shaped conidia. These spores gain the surface of other 
silkworms or butterflies by contact or by air transmission, and 
germinate, sending threads into their bodies. Sickle-shaped 
spores are produced from these inside the body, and these grow 
out into threads, forming a mycelial network throughout the body 
of the victim and causing its death. It is possible that several 
different species of molds may be concerned in the causation: 
of muscardine. 

The fungus is of interest because it was probably the first 


244 SPECIFIC MICRO-ORGANISMS 


mold to be recognized as a cause of disease, and also because 
it is an example of a large group of fungi which attack various 
insects. The disease muscardine is, moreover, one of consider- 
able importance to the silk industry. 

Oidium Lactis.—Oidium lactis is very widely distributed and is 
almost always present in milk and milk products, and in brewer’s 
and baker’s yeast, and it is an especially prominent organism in 


Fic. 96.—Oidium lactis. a, b, Dichotomous branching of growing hyphe; ¢, d, 
gz “simple chains of oidia breaking through substratum at dotted line x—y, dotted por- 
tions submerged; e, f, chains of oidia from a branching outgrowth of a submerged 
cell; h branching chain of oidia; k, 1, m, n, 0, p, s, types of germination of oidia under 
varying conditions; #, diagram of a portion of a colony showing habit of Oidium 
lactis as seen in culture media. (From Bull. 82, Bur. Animal Industry, U.S. Dept. 
Agr.) 


the further fermentation of, acid substances, such as sauerkraut, 
sour milk and cheese. The organism is especially important in 
the ripening of Camembert cheese. It grows well on ordinary 
nutrient gelatin. The colony consists of a loosely woven, white 
network of septate, branched and anastomosing threads, chiefly 


MOLDS AND YEASTS AND DISEASES’ CAUSED BY THEM 245 


in the substratum but also extending into the air. The peripheral 
threads are divided by septa to form chains of oval or spherical 
conidia. 

This mold may be readily obtained for study by making plate 
cultures from compressed yeast. 


Fic. 97.—Oidium albicans. A deep colony on a plate culture of the liquefying 
variety, showing chlamydospores. (After Plaut.) 


Oidium Albicans (Monilia Candida.)—The thrush fungus 
was discovered by von Langenbeck in 183g and by Berg in 1841, 
but the popular recognition of a relation between this disease and 
a mold seems to have preceded this discovery by many years. 
Berg (1841) transferred the fungus from cases of thrush to healthy 


246 SPECIFIC MICRO-ORGANISMS 


children with positive results. His work was confirmed by numer- 
ous other investigators (1842-43). Robin (1847) accurately 
described the parasite, with illustrations, classed it as an oidium, 
and gave it the name Oidium albicans (1853). Grawitz (1877) 
obtained the first pure cultures and-successfully inoculated rab- 
bits and puppies with them. 

In the throat lesion as well as in cultures the organism con- 
sists of mycelial threads and oval yeast-like cells. It grows read- 
ily on various artificial media and the appearance of the growth is 
quite variable, not only because of the proportional relation be- 
tween the oval cells and the threads, but also in pigmentation 
and in density of growth. Two yarieties, one liquefying gelatin 


[= 


and producing large (5m) oval conidia, and the other failing to 
liquefy gelatin and producing small (2.54) spherical conidia are 
distinguished. 

Thrush is most common on the buccal mucous membrane of 
young infants, but it also occurs on the vaginal mucosa of preg- 
nant women, and it may attack others when weakened by dis- 
ease, especially diabetics. The disease also occurs naturally in 
birds, calves and foals. The threads of the mold penetrate the 
squamous epithelium and even enter the subepithelial tissue, 
sometimes penetrating blood-vessels and giving rise to metas- 
tases. It results in‘death in about 20 per cent of the cases in 
infants. The predisposing digestive disorder or other primary 
disease is however, usually more important than the thrush, and 
demands first consideration in treatment. The thrush lesion may 
be carefully removed with a soft swab and the eroded area treated 
with silver nitrate, o.1 per cent. Generalization of the disease 


MOLDS AND YEASTS AND DISEASES CAUSED BY THEM 247 


is rare, but several cases have been observed. Inoculaticn of 
animals (mice, guinea-pigs, puppies, rabbits) is sometimes success- 
ful, and generalized thrush has followed intravenous injection of 
young rabbits. The fungus seems to exert some poisonous action, 
in addition to the mechanical effect upon the tissues. 


é _ 


Fic. 99.—Scutulum of favus on the arm of aman. (After Plaut.) 


Monilia Psilosis——Ashford! has found a yeast-like organism 
on the tongue and in the feces of persons suffering from sprue and 
in collaboration with Michel has demonstrated the presence of 
a complement-fixation reaction between the blood of sprue pa- 
tients and an antigen prepared from cultures of this organism. 
‘The cultures are made on acid glucose agar or on Sabouraud’s 
medium. Sprue is a chronic disease characterized by recurrent 
attacks of diarrhea, with foamy, whitish and bulky stools, progres- 
sive emaciation and weakness. The subject requires further 
investigation before the causal relation of Monilia psilosis to 
. the disease can be accepted as established. 

Achorion Schoenleinii—The fungus of favus was discovered by 
Schoenlein in the skin lesions of this disease in 1839, two years 
after the recognition of Botrytis bassiana as the cause of mus- 
cardine. Remak in 1845 grew the mold on slices of apple and 

_ Ashford, Amer. Journ. Med. Sciences, 1916, 151,p. 520; ibid., 1917, 154,P-157 


248 SPECIFIC MICRO-ORGANISMS 


successfully inoculated his skin with these cultures. He name 

the organism Achorion schoenleinii. In the lesion of favus the 
threads of the fungus are found growing in the horny layer of the 
epidermis, usually about a hair, and giving rise to a dry, circular, 
yellow crust with depressed center, the ““Scutulum.”” By macerat- 
ing this crust in 50 per cent antiformin the elements of the mold 
are made clearly visible under the microscope. In the center of 
the lesion are doubly contoured oval or rectangular conidia, 3 


Fic. 100.—Typical scutulum of favus in a mouse. (After Plaut.) - 


to 84 by 3 to 4u, single and in chains. The mycelial threads 
are indistinguishable in the center, but are seen at the periphery 
as tubes of very irregular width, refractive with granular proto- 
plasm, often branched or knobbed at the end. The scutulum 
in its interior is a pure culture of the mold, entirely free from other 
organisms. The mold also grows in the interior of the hair shaft, 
and by macerating the hair in alkali the fungus may be demon- 
strated microscopically. 

Cultures may be obtained upon various media. Plaut recom- 
mends a medium containing pepton 1 to 2 per cent, glycerin 


MOLDS AND YEASTS AND DISEASES CAUSED BY THEM 249 


0.5 per cent, salt 0.5 per cent and agar 2 per cent, without meat 
extractives or any addition of alkali. The cultures are incubated 
at 30° C. Mycelial threads and numerous conidia are produced. 


Fig. 101.—Achorion schoenleinii, Colony developing from a favusscale. End, en- 
doconidia on submerged hyphe. Ect, ectospores on aerial hyphe. (After Plaut.) 


Inoculation into the epidermis of mice or onto the human 
skin gives rise to typical lesions. Intravenous injection into 
rabbits is usually followed by a pseudo-tuberculosis in the lungs, 
sometimes fatal. Similar skin lesions occur naturally in various 
animals, and the molds there present are very similar to, if not 


250 SPECIFIC MICRO-ORGANISMS 


specifically identical with, Achorion schoenleinii. The exact 
relationships of the parasites are not very fully settled as. yet. 

Microsporon Audouini.—This mold is found growing in the 
hair-shaft in alopecia areata. If the hair be pulled out it breaks 
near the lower end and the oval 
conidia and jointed threads of the 
parasite may be demonstrated by 
macerating this broken end. The 
disease is very contagious, chronic 
and resistant to treatment, but 
proceeds without inflammation or 
subjective symptoms, the conspic- 
uous sign being loss of the hair. 
Cultures grow slowly and are snow 
white. Animal inoculation is rarely 
successful. 

Microsporon Furfur.—This mold 
is found in the superficial layer. of 
the skin in pityriasis versicolor, as 
short thick hyphe 3 to 4» wide by 
7 to 13 long, together with abundant 
doubly contoured single conidia. 
Pityriasis versicolor occurs most fre- 
quently on the skin of the chest and 
is one of the commonest affections 
of the skin. 

Tricophyton Acuminatum.—The 


Fic. 102.—Sporotrichum 
schencki. Cultures on the glu- 


cose-pepton agar of Sabouraud. mold invades the hair shaft and 


(After Gougerot.) « 
i causes it to break off close to the 


surface of the skin. In such a hair long chains of oval cells of 
the parasite may be seen. The parasite also attacks the skin and 
produces ringworm. Several other species of tricophyton are 
distinguished. These parasites are concerned in the causation of 
barber’s itch, eczema marginatum, tinea cruris, and other skin 
affections of this type.. 


MOLDS AND YEASTS AND DISEASES CAUSED BY THEM 251 


Sporotrichum Schencki—Schenck, at Baltimore in 1808, de- 
scribed this parasitic mold which he found in the lesions of a 


6 


=a 


5 


Fic. 103.—Sporotrichum schenki. Various forms of mycelium with and without. 
conidia. (After Gougerot.) 


peculiar disease, beginning as a localized ulcer, with later involve- 
ment of the neighboring lymph glands, in which cold abscesses 


252 : SPECIFIC MICRO-ORGANISMS 


formed and opened to the exterior. A second similar case was 
described by Hektoen and Perkins. Ruediger’ has. reported a 
large series of cases of sporotrichosis and the references to Ameri- 
can literature will be found in his paper. The organisms are not 
readily found in the pus by microscopic examination and seem 
to exist there only as conidia. In cultures a branching mycelium 
with clusters of conidia is produced. Dogs are susceptible to 
inoculation. : 


Fic. 104.—Doubly contoured organisms found in oidiomycosis (blastomycosis). 
(From Buschke after Hyde and Montgomery.) 


Sporotrichium Beurmanii—De Beurmann and Ramond at 
Paris in 1903 found this parasite in a case of lymphangitis. It 
seems to be different from the organism described by Schenck 
but may ultimately prove to be the same species. 

Cryptococcus Gilchristi—Doubly contoured yeast-like cells 
which multiply by budding in human tissues, were first discovered 
by Busse and Buschke? in 1894, in a case presenting abscesses in 


1 Journ. Infect. Diseases, 1912, Vol. XI, pp. 193-206. 
2 Deutsch. med. Wochenschr., 1895, Nr. 3. 


MOLDS AND YEASTS AND DISEASES CAUSED BY THEM 253 


the bones and internal organs together with lesions of the skin. 
They obtained cultures of the organism and classed it as a yeast. 
About the same time Gilchrist! independently observed similar 
organisms in cases of dermatitis at Baltimore. The organisms 
have been most thoroughly studied by Ricketts.2 Most of the 
cases have been observed in the United States, particularly at 
Baltimore and at Chicago. The disease is designated as oidiomy- 
cosis, blastomycosis and blastomycetic dermatitis. It exists 
most commonly as a chronic purulent dermatitis but infection of 
the pericardium and of the meninges with these organisms and 
even generalized blastermycosis has been reported.* In the earlier 
literature this organism was often confused with Coccidioides 
immitis. On glucose agar, the parasites usually grow without 
difficulty and the growth resembles that of an oidium, often with 
abundant aérial hyphe. Inoculation of guinea-pigs with pus 
or with cultures is usually followed by formation of abscesses in 
which the typical spherical or ovoid parasites may be found. 
Further investigations are required to determine the specific 
relationships of the parasites found in different cases. 


1 Gilchrist: Johns Hopkins Hosp. Rept., Vol. I, p. 209, 1896. 

2 Journ. Med. Research, Vol. VI, No. 3. 

3Sihler, Peppard and Cox, Case of systemic blastomycosis, Journal-Lancet 
(Minneapolis) 1917, 37, DP. 253- 


CHAPTER XV 
TRICHOMYCETES 


The trichomycetes or higher bacteria are intermediate in 
morphological characters between the molds and the lower bac- 
teria. They resemble the molds in the formation of long threads, 
sometimes branching and interlacing to produce a network, and 
in the formation of oval or spherical conidia constricted off from 
the ends of the threads. They resemble the lower bacteria in 
their small transverse diameter, the delicacy of their structure 
and their mode of life. Petruschy' recognizes four genera, 
Actinomyces, Streptothrix (Nocardia), Cladothrix and Leptothrix. 

Actinomyces Bovis.—Bollinger in 1877 studied the lumpy-: 
jaw disease of cattle and described this parasite which occurs 
in the lesions. Israel, in the following year found the organism 
in granulomatous lesions in man. The infection also occurs in 
horses, sheep, swine and dogs. In the tissues and in the purulent 
discharge from the lesions, the organism occurs in small yellowish 
masses, sometimes visible to the naked eye but usually smaller 
(10 to 200 in diameter). Such a mass is a single colony of the 
parasite or a conglomerate of several colonies. The colony is a 
dense network of threads in the center, with radially arranged 
threads about the periphery, most of the latter being swollen, 
club-shaped, at their free ends. Spherical bodies may also be 
present, but whether these are conidia or degeneration forms of 
the parasite is uncertain. The organism is Gram-positive. 

Inoculation of pus or bits of tissue containing the parasite 
from one animal into another usually fails to transmit the disease, 
although positive results have been obtained in a few instances. 
Attempts at culture have failed also in many instances, and the 


1 Kolle and Wassermann: Handbuch, 1912, Vol. V, p. 270. 
254 


TRICHOM YCETES 255 


difficulty here seems to depend in part upon the oxygen require- 
ments of the organism. The material for culture should be 
obtained from uncontaminated tissue containing the fungus. 
If this is impossible, the granule of actinomyces should be washed 
in several changes of sterile salt solution, then crushed between 
sterile glass slides or, better, ground up in a sterile mortar with 
a small amount of sterile sand. A series of dilution cultures 
should then be made in tall tubes of melted, glucose agar cooled to 
45° C., the tubes chilled in cold water and incubated at 37° C. 


Fic. 105.—Actinomyces bovis. The ray-fungus from cow. (Diagrammatic.) 


Colonies of the fungus may be expected to develop some distance 
below the surface of the agar. Wolf and Israel were able to re- 
produce the disease in animals (rabbits and guinea-pigs) by the 
inoculation of pure cultures. More recently Harbitz and Gron- 
dahl’ isolated twenty-seven strains of actinomyces, but their 
inoculation experiments were wholly negative. It would appear 
that other factors are essential to the development of actinomy-. 
cosis in addition to the inoculation of the specific parasite. Many 
authors regard the presence of bits of straw or sharp grains in 
wounds of the mucous membrane of the mouth or pharynx as 
important elements in predisposing to infection with actinomyces. 


1 Amer. Journ. Med. Sciences, 1911, Vol. CXLII, pp. 386-305. 


2 56 a SPECIFIC MICRO-ORGANISMS 


The disease shows little or no tendency to be transmitted from 
animal to animal in a herd. Several varieties of actinomyces 
have been described, and possibly more than one species will 
eventually be recognized. 

Streptothrix Madura.—Kanthack (1892) and Gemy and 
Vincent (1892) discovered the fine mycelial threads in pus from 
cases of Madura foot. Granules about-the size of a pin-head occur 
in the pus, and under the microscope these are found to consist of 
a network of threads 1 to 1.5u in thickness, arranged radially 
at the periphery and presenting somewhat swollen ends. These 
granules are white in some cases, yellow, red and black in others. 
The nature of the disease seems to be the same in all cases, but 
the micro-organisms are apparently not the same, that found in 
the black variety probably representing a distinct species. Cul- 
tures may be obtained by inoculating the pus, collected without 
contamination, into several flasks of sterilized hay infusion, and 
shaking daily to insure abundant oxygen supply. It also grows 
upon other media. Gelatin is not liquefied. The growth is made 
up of interwoven, slender branching threads about 1 y in thickness. 
Spores (conidia) capable of resisting a temperature of 75° C. for 
five minutes are produced at the surface of the culture. Inocu- 
lation of animals usually gives negative results, but Musgrave 
and Klegg! have succeeded in infecting monkeys. 

The disease, Mycetoma or Madura foot, is a localized chronic 
inflammation, almost painless, and usually involving the foot, 
the hand or some exposed portion of the body. The disease 
involves the tissues by direct extension, attacking the bones as well 
as the soft tissues. It usually remains localized to one extremity. 

The black variety of Madura foot is due to a different organ- 
ism, the threads of which are 3 to 8u in thickness.? This organ- 
ism seems to be an aspergillus, and has been named Madurella 
mycetort. 


1 Philippine Journ. of Science, 1907, Vol. II, pp. 477-512; A complete‘ bibli- 
ography by Polk is included. 
2 Wright: Journ. of Exp. Medicine, Vol. III, pp. 421-433. 


TRICHOMYCETES 257 


Streptothrix putorii (Nocardia putorii)—Dick and Tunnicliff? 
have found this organism in a case of fever following the bite 
of a weasel. Somewhat similar organisms have been found in 
fever following rat bites: The usual cause of rat bite fever is, 
however, evidently a spirochete, Spirocheta (morsus) muris. 

Streptothrices have also been found in abscesses of the brain 
and in chronic disease of the lung clinically resembling tuberculosis 
in man. Many of them are Gram-positive and some are rela- 
tively acid-proof when stained with carbol-fuchsin. Such acid- 
proof forms are common in the feces of cattle where short 
segments of them may be mistaken for tubercle bacilli. Organ- 
isms of this type are very abundant in the soil, which is 
doubtless their natural habitat. - 

Cladothrix.—The cladothrix forms resemble the strepto- 
thrices very closely but the cells of the threads do not branch. 
The apparent branching of the threads is explained as due to a 
transverse division of the thread with continuing growth of the 
one free end. which-pushes out beyond the other, giving rise to 
the appearance of branching or so-called “false branching.” 
Organisms of this type have been described as occurring in ab- 
scesses of the brain and in other parts of the body. The dis- 
tinction from streptothrix has not always been clearly made. 

Leptothrix Buccalis.—This is a normal inhabitant of the mouth 
cavity. It consists of slender filaments which do not branch. 
The organism has been found in abundance in small white patches 
on the tonsils, where it sometimes causes a very chronic but mild 
inflammation. Artificial culture of the organism ordinarily 
results in failure. Arustamoff? appears to have obtained it on 
a neutral or acid agar inoculated with leptothrix from urine. 


1 Dick and Tunnicliff: Journ. Infectious Diseases, 1918, 23, p. 183. 
2 Kolle and Wassermann: Handbuch, ror2, Bd. V, S. 290. 


i? 


CHAPTER XVI 


THE COCCACEZ AND THEIR PARASITIC RELATION- 
SHIPS 


Diplococcus Gonorrhez (Neisseria Gonorrhez).—The gono- 
coccus was discovered by Neisser! in 1879 in the discharge of acute 
urethritis and he recognized its probable causal relationship to 
the disease. Cultures were first obtained by Bumm? in 1885 and 
he proved the relationship by inoculating the human urethra with 
his cultures. The organism naturally lives and multiplies only 
in the human body and is the cause of gonorrhea and many of its 
complicating inflammations. ~ 

The gonococcus is found in both the serum and the poly- 
nuclear cells of the purulent discharge, usually in pairs with the 
adjacent surfaces flattened. The long diameter of the pair is 
about 1.254. It stains readily, best perhaps with Léffler’s methy- 
lene-blue. It is decolorized when stained by Grams’ method, a 
fact of great importance in the quick recognition of the organism. 
The staining procedure has to be carefully carried out and a 
beginner should practice upon cultures of the gonococcus and upon 
samples of gonorrheal pus and staphylococcus pus before placing 
too much reliance upon the appearance of his Gram-stained prepa- 
ration. The teaction to the Gram stain, together with the re- 
markably characteristic appearance of the pus cell full of diplo- 
cocci are usually sufficient for the recognition of the organism in 
acute urethritis. 

Cultures of the gonococcus were obtained by Bumm on coagu- 
lated human blood serum. Wertheim? employed serum agar 

' Neisser: Centralbl. f. d. med. Wissenschaft, 1879, Bd. XVII, S. 497-500. 

?Bumm: Deutsche med. Wochenschr., 1885, Bd. II, S. 910 and QIl. 


3 Deutsche med. Wochenschr., 1891, Bd. XVI, S. 958; S. 1351 and 1352. 
258 


- 


COCCACEZ AND THEIR PARASITIC RELATIONSHIPS 259 


made by mixing human blocd serum at 40° C., one part, with 
ordinary nutrient agar melted and cooled to 40° C., two parts. 
_ The medium may be inclined in tubes or may be employed for 
plating. Human ascitic fluid or hydrocele fluid is just as good 
as blood serum. A large drop of pus from an acute urethritis 
should be mixed with 2 to 3 c.c. of serum or ascitic fluid in a 
test-tube and, from this, dilutions made to a second and a third 
tube. The contents of a tube of agar (5 to 6 c.c.), previously 
melted and cooled to about .40° C., is then added to each tube of 


Fic. 106.—Gonococci and pus-cells. X1000. 


serum, mixed thoroughly and poured into Petri dishes to solidify. 
At 37° C., colonies appear within 24 hours and at the end of this 
time measure about 1 mm. in diameter. The colony is circular, 
grayish-blue and transparent and of a mucoid consistency. — 
The individual cocci disintegrate rapidly, even within the first 
24 hours at the center of the colony, and for microscopic study 
simple staining and staining by Gram’s method, cultures 5 to 10 
hours old are recommended. Even under favorable conditions 
the gonococcus ordinarily dies out in the culture tube in about a 
week, although exceptionally it may survive for three weeks. 


260 SPECIFIC MICRO-ORGANISMS 


It should be transplanted every few days and a large quantity 
of growth must be transferred. When transplanted from vigor- 
ous cultures to plain agar the gonococcus grows for a few days, 
but it cannot be successfully propagated for any length of time 
on ordinary media. 

The gonococcus is very sensitive to drying and to tempera- 
tures above 40° C. It is usually impossible to recover it from 
dried pus, but in moist material it may live for 1 to 24 hours. 
The organism is easily killed by chemicals germicides, of which 
silver nitrate is probably the most effective. . 

Inoculation of animals in the urethra or on the conjuctiva 
is without result. Intraperitoneal injection of cultures into 
-white mice or’ guinea-pigs usually kills the animals in 24 hours 
and the gonococci can be recovered from the peritoneal fluid 
and the heart’s blood. These effects seem to be due to toxins of 
the injected material rather than actual infection. The specific 
poisons seem to be intracellular and set free upon disintegration 
of the organism. The poison withstands heating to 100° C. for 
hours. Inoculation of the human urethra with cultures of the 
gonococcus has been repeatedly done and has resulted nearly al- 
ways in the production of typical gonorrhea. : 

Gonorrhea has been recognized as a contagious disease since 
the dawn of history. The most important forms are (1) urethritis 
with tendency to extension in the female to the cervix uteri, ovi- 
ducts and peritoneum, and in the male to the prostate, seminal 
vesicles and epididymis, and in both sexes to the blood stream, 
heart valves and joints; (2) conjunctivitis and keratitis leading to 
scarring of the cornea and permanent blindness; (3) vulvo-vaginitis 
in girl babies, an exceedingly contagious disease, especially in 
hospital wards. The disease tends to become chronic and eventu- 
ally latent, that is, the symptoms subside but the, micro-organisms 
remain alive in certain locations, such as the prostate in the male 
and the cervix uteri in the female. The acute inflammation may 
be followed by scars resulting in strictures of the urethra or occlu- 
sion of the epididymis. In the female, pyosalpinx is a not unusual 


COCCACEH AND THEIR PARASITIC RELATIONSHIPS 261 


complication. Secondary infection with staphylococci is common 
in chronic gonorrhea. 

Specific diagnosis by finding gonococci usually presents no 
difficulties in acute inflammations of the genital tract, in which 
the characteristic groups of Gram-negative intracellular diplococci 
are practically diagnostic. In chronic cases and in extra-genital 
inflammations the diagnosis presents greater difficulty. Both 
microscopic and cultural examinations should be made and if 
negative they should be repeated many times. Even repeated 
failure to find the gonococcus by these methods does not justify 
the positive assertion that it is absent. Specific diagnosis by 
the method of complement fixation has been developed by Sch- 
wartz and McNeill.!. The antigen is prepared from several cul- 
ture strains of the gonococcus and in all other respects the test 
is similar to the Wassermann test for syphilis. Irons? has em- 
ployed a cutaneous test, using a glycerin extract of gonococci. 
The technic is similar to that of the von Pirquet test for 
tuberculosis. 

The prevalence of gonorrhea throughout the civilized world 
is much greater than has been popularly supposed. Erb, in a 
study of 2000 males among private patients of the middle and better 
classes, found a history of gonorrhea in 50 per cent. Many 
other students of the disease disagree with Erb, regarding his 
figures as much too low. The large mass of statistics obtained by 
examination of recruits for war service in 1917 and 1918 indicates 
that approximately 2 per cent of men in the age period 21 to 31 
years in the United States are afflicted with recognizable gonorrhea 
at any one time. Among women in German obstetrical hospitals, 
largely from the poorer class, gonorrhea is present in ro to 30 per 
cent. The danger to the eyes of the new-born infant is now over- 
come by the use of silver nitrate in the eyes when they are first 
cleansed. The general prevention and restriction of gonorrheal 
infection is engaging more and more the serious attention of 


1 Amer. Journ. med. Sciences, 1912, Vol. CXLIV, pp. 815-826. 
2 Journ. Infec. Diseases, 1912, Vol. XI, PP. 77-93- 


262 SPECIFIC MICRO-ORGANISMS 


thoughtful citizens, and it is already recognized as a sanitary 
problem of the first magnitude. 

Diplococcus Meningitidis (Neisseria Intracellularis).— Weich- 
selbaum in 1887 examined the cerebrospinal fluid in six sporadic 
cases of meningitis and found in all of them a very definite Gram- 
negative intracellular diplococcus, the meningococcus. He ob- 
tained cultures but his animal inoculations all gave ‘negative 
results. Jaeger in 1895 seems to have found a similar organism in 
fourteen, cases of epidemic meningitis and Huebner in 1896 ap- 
parently found it in five cases. The cultural work of these authors 
seems to be unreliable as their cultures were Gram-positive. 
More conclusive confirmation of the relation of this organism to 
epidemic meningitis was furnished by Councilman, Mallory and 
Wright! in 1918. 

The meningococcus is found in the bodies of patients suffering 
from meningitis, occasionally on the nasal mucous membrane 
of healthy persons and of cases of rhinitis, and very rarely in 
other situations. In cerebrospinal meningitis the organism is 
present in the cerebrospinal fluid, in the meninges, often on the 
nasal and pharyngeal mucous membrane, sometimes in the 
blood and on the conjunctive, and rarely in the urethra, where 
it may be mistaken for the gonococcus. It is usually found 
without difficulty. in the cerebrospinal fluid in the first few days 
of the disease, but may be very difficult to find at a later stage. 

The organism is found for the most part inside polynuclear 
leukocytes and in its form, size, arrangement and behavior to 
the Gram-stain resembles very closely the gonococcus. The 
outline of the cocci is often somewhat hazy, suggesting possible 
disintegration, and this sometimes makes their recognition 
somewhat difficult in microscopic preparations of cerebrospinal 
fluid. Cultures may be made on ascitic-fluid agar or blood agar, 
upon which small dew-drop colonies appear in 24 hours at 37° C. 
A better medium is obtained by laking human blood or rabbit’s 


1 Report of the Mass. Bd. of Health on Epidemic Cerebrospinal Meningitis, 
etc., Boston, 1898. 


COCCACEZ AND THEIR PARASITIC RELATIONSHIPS 263 


blood with distilled water and adding this to melted glucose agar 
in a quantity sufficient to give it a pink tint. The color of blood 
is.unaltered by the growth. Cultures may be obtained on Loffler’s 
blood serum, although this medium is not very satisfactory for 
this purpose. The meningococcus grows more luxuriantly than 
the gonococcus, as a rule, and adapts itself more readily to growth 
on ordinary media, but its cells disintegrate rapidly in the colony, 
which is viscid. In nearly every respect it resembles very closely 
the gonococcus. 

Intraperitoneal inoculation of white mice and of guinea-pigs 
usually results in fatal peritonitis and the organism can be recov- 
ered from the heart’s blood. Intraspinal inoculation of monkeys 
with large doses causes typical meningitis with symptoms similar 
to those of the disease in man. In man the disease is undoubtedly 
transmitted very largely by coccus-carriers, healthy people or 
people with slight pharyngitis or rhinitis, who carry the virus on 
their mucous membranes and distribute it. 

Several serologically different types of meningococci are 
recognized and for the specific recognition of the meningococcus 
by the agglutination test, it is advisable to employ polyvalent 
serum as well as the various mono-valent sera in dilutions of 1x 
to 100 and 1 to 200. Suspensions of living cultures grown on plain 
agar or on serum agar, if necessary, are mixed with the serum and 
the tubes are incubated 16 hours at 55° C. A control tube of 
normal serum should be included in the test. 

Ordinarily, specific agglutination and type determination may 
be neglected in the recognition of meningococci found in the 
cerebro-spinal fluid. -When isolated from the pharynx, bronchi 
or lungs, confusion with other organisms is more probable and, in 
these instances, agglutination tests are required. Bacteriologists 
undertaking this work should consult the paper of Flexner! and 
the literature there cited, especially the Gordon report. 

Antimeningococcus serum is prepared by immunizing horses 


1 Flexner, S.: Mode of infection, mears of prevention and specific treatment 
of epidemic meningitis, Journ. Amer. Med. Assn., 1917, 69, Pp. 639, P- 721, P- 817. 


264 SPECIFIC MICRO-ORGANISMS 


with a mixture of many typical and atypical meningococcus 
cultures injected subcutaneously. At first the cultures are 
killed by heat before injection, and only one or two loopfuls are 
given. The dose is increased and repeated: every 8 to 10 days 
until the growth on two Petri dishes is being injected. Living 
cultures are then given, and finally old cultures: which have 
disintegrated are also used. The serum is used after the horse 
has been treated for 8 to 10 months. Jochmann showed that 
the subcutaneous injection of the serum is without effect upon 
meningitis in monkeys but that when introduced into the spinal 
canal is specifically curative. Flexner! and his co-workers have 
studied this very fully and there can no longer be any question 
of the value of the serum in the treatment of meningococcus 
meningitis. 

Cerebrospinal fluid is obtained by Quincke’s puncture. For 
children a needle 4 cm. long and with a lumen of 1 mm. is intro- 
duced in the medium line directly forward so as to enter the 
spinal canal between the second and third or the third and fourth 
lumbar vertebra. From 20 to 50 c.c. of fluid may be withdrawn 
if it comes away under pressure, and then the curative serum 
is injected through the same needle. The fluid withdrawn should. 
be examined to establish the presence of meningitis and its variety. 
In general the examination includes a macroscopic examination 
and description of the appearance of the sample, ‘a microscopical 
numerical count of the cells present, chemical examination of 
the cell-free fluid for excessive protein’ content, microscopic and 
cultural examination of the sediment for bacteria and of the 


1 Flexner: Harbin lectures. Journ. State Medicine, 1912, Vol. XX, pp. 257-270. 

2 Noguchi’s test: To 0.5 c.c. of blood-free fluid add 1 c.c. ro per cent butyric 
acid, boil; add 0.2 c.c. normal NaOH and boil again. Set aside to cool. A floc- 
culent precipitate indicates an increase in the globulin content. 

Pandy’s test: Add 1 drop of the spinal fluid to 1 c.c. of a saturated aqueous 
solution of carbolic acid. The immediate formation of a bluish-white Ting or cloud 
indicates an increased protein content. 

Ross-Jones test: Layer equal quantities of spinal fluid and ammonium sulphate 
solution, saturated by boiling. A white ring indicates abnormal globulin content. 


COCCACEZ AND THEIR PARASITIC RELATIONSHIPS « 265 


filmy clot which may form after standing an hour or so for tubercle 
bacilli, and sometimes it includes the Wassermann reaction. 
In meningococcus meningitis the cell count is generally above 
100 per cu. mm., and most of the cells are polynuclear leukocytes. 
Within these cells the meningococci may or may not be found. In 
case of doubt, plate cultures on blood-agar and ascitic-fluid agar 
should be made. The recognition of a Gram-negative intracellular 


Fic. 107.—Meningococcus in spinal fluid. (After Hiss and Zinsser.) 


diplococcus in the fluid is sufficient for a tentative diagnosis, and 
the appearance of characteristic colonies on the plates may be~- 
considered conclusive. 

Diplococcus (Micrococcus) Catarrhalis.——This organism is 
commonly present on the mucous membrane of the upper air 
passages, especially in catarrhal inflammations. It is usually 
seen as a Gram-negative intracellular diplococcus not to be 


266 > SPECIFIC MICRO-ORGANISMS 


distinguished microscopically from the meningococcus or gono- 
coccus. In examining material from the air passages this organ- 
ism has to be considered. It is readily distinguished by cultural 
methods. On ascitic-fluid agar the colony is dry and brittle, 
quite different from the meningococcus or gonococcus. Further- 
more, it grows readily at once on ordinary agar. 

Diplococcus Pneumoniz.—Sternberg in 1880 injected the 
saliva of healthy persons into rabbits and produced a rapidly 
fatal bacteremia with abundant lance-shaped diplococci in the 
blood and internal organs of the animal. Pasteur, independently 
and at about the same time, injected the saliva of a boy suffering 
from rabies into rabbits with a similar result. The organism 
was spoken of as the diplococcus of sputum septicemia or the 
septicemic microbe of saliva. Koch in 1881 demonstrated the 
organism microscopically in sections of lung. Friedlaender 
(1882-1884) found the organism microscopically in a large number 
of cases of pneumonia and accurately described its form, the 
capsules and staining properties. His cultures, however, which 
were made on gelatin at.room temperature, brought to develop- 
ment not the pneumococcus but a wholly different organism which 
he believed to be identical with it, Friedlaender’s pneumobacillus. 
A. Fraenkel obtained the first undoubted pure cultures on solidified 
blood serum, proved the identity of the organism in pneumonia 
with that of normal saliva seen by Sternberg and Pasteur, and 
distinguished it absolutely from the pneumobacillus of Fried- 
laender. He also succeeded in producing typical pneumonia by 
injecting cultures of moderate virulence intravenously into rabbits. 

The pneumococcus is somewhat variable in form. In the 
animal body it occurs.in pairs of lance-shaped individuals with the 
points directed away from each other, and the pair is surrounded 
by a thick gelatinous capsule.1 The organism is always Gram- 


1In demonstrating the capsules, the method of Hiss gives excellent results. 
Spread some blood or tissue juice on a cover-glass and as soonas the film of moisture 
has disappeared, fix the preparation by heat. Then stain with hot aqueous gentian 
violet and wash off the dye with a 20. per cent solution of copper sulphate. Examine 
in the copper solution. Blot the preparation, dry it in air and mount in balsam. 


COCCACEA AND THEIR, PARASITIC RELATIONSHIPS 267 


positive. In cultures the capsules are less well developed and 
often cannot be demonstrated at all. The individuals are often 
less pointed and frequently resemble short bacilli in form. They — 
may remain attached together in chains of six to eight cells. 
Cultures may be obtained on ordinary media but they are 
prone to die out quickly. Blood-agar, serum agar or ascitic-fluid 
agar are the best solid media, but even with these weekly trans- 
plantation is usually necessary. Broth to which serum or ascitic 
fluid has been added forms an excellent medium. There is prac- 


Fic. 108. —Pneumococcus, showing capsule, from pleuritic fluid of infected rabbit, 
. stained by second method of Hiss. 


tically no growth below 25° C. On blood agar, the colony is 
surrounded by a zone of greenish discoloration, a character of 
great value in the early recognition of the pneumococcus isolated 
from the body. The virulence of the microbe diminishes very 
rapidly in artificial culture. Virulent material is best kept in 
stock by preserving in a desiccator dried blood taken from a 
rabbit dead of pneumococcus infection. The fluid blood may also 
be kept in sealed capillaries in the refrigerator. By these methods 
the virulence may be preserved for months. Rabbits, mice and . 
young rats are the most susceptible animals. 


268 SPECIFIC MICRO-ORGANISMS 


The pneumococcus is the microbic agent in from 80 to 95 
per cent of cases of acute lobar pneumonia. It also occurs in 
otitis media, mastoiditis, meningitis, peritonitis and arthritis. 
Its presence is usually associated with a fibrino-purulent exudate 
In severe pneumonia it is often present in the circulating blood. 

Pneumonia, or inflammation of the lungs, may be caused 
by a great variety of organisms, the tubercle bacillus, the pneu- 
mobacillus of Friedlaender, the streptococcus, the typhoid 
bacillus and many others. Typical lobar pneumonia, however, 
a disease characterized by a definite sequence of pathological 
changes in the lung and by a rather typical clinical course, is © 
rarely caused by any organism other than Diplococcus pneumonie. 
This is a very frequent disease in adults and doubtless the most 
frequent cause of death in persons over 50 years of age. 

The nature of the poisons produced by the pneumococcus 
is not definitely known. When killed by heat, the dead germ 
substance is not very toxic. One very remarkable property of 
the organism is its susceptibility to the action of bile and solutions 
of bile salts. These cause a complete and prompt solution of 
suspensions of pneumococci. Cole! has shown that a powerful 
poison is set free by this disintegration of pneumococci, the 
toxic action of which resembles that seen in the phenomenon 
of anaphylaxis. 

It has been possible to induce a high degree of immunity in 
horses, and the serum of these animals is protective and curative 
in animal experiments. By use of such serum a large number of 
serologically different strains of pneumococci have been recognized. 
Cole and his associates have found that a considerable proportion 
of labor pneumonia in New York City is caused by one of these 
serological types, which they have designated as Group I, and they 
have been able to produce an effective anti-serum against these 
strains. A second, somewhat less homogeneous collection of 
strains is characterized by specific reaction with another immune 


1 Cole: Journ. Exp. Med., 1912, Vol. XVI, pp. 644-664; Harvey Lectures, 1913- 
14, Lippincott, p. 85. This paper gives references to literature. 


COCCACEH AND THEIR PARASITIC RELATIONSHIPS 269 . 


serum and these types are designated as Group II. Group III 
includes the morphologically different Pnueumococcus mucosus, 
characterized by its large capsules, the viscid nature of its colo- 
nies on culture media and of the peritoneal exudate which it 
produces in the mouse. The remaining strains of pneumococci 
are placed in a heterogeneous Group IV. Infections with pneu- 
mococci of Group I are favorably influenced by the injection of 
Type I serum. The Type II serum appears to be of some value 
in treating infections with pneumococci of Group II. No thera- 
peutic serum has been produced for Group III and the sera 
obtained in Group IV are potent against only the particular strain 
employed for immunization of the animal. Determination of 
the Group of the infecting pneumococcus should, therefore, pre- 
cede the therapeutic use of the serum. 

For type determination! the sputum should be obtained from 
the deeper air passages under immediate supervision of the bac- 
teriologist and should be examined immediately. Microscopic 
examination of preparations stained by (1) Gram’s method, (2) 
Ziehl-Neelsen method and (3) Hiss capsule stain should be done. 
Pneumococcus mucosus shows wide capsules in both the Gram and 
the Hiss preparations. A piece of sputum, volume about 0.2 
c.c., is washed through four changes of salt solution in Petri dishes, 
placed in a mortar, ground up and emulsified in about 1 c.c. of 
salt solution, which is added drop by drop. About 0.5 c.c. of 
this suspension is injected intraperitoneally into a white mouse. 
The common sputum organisms which grow in the peritoneal 
cavity of the mouse are the pneumococcus, the influenza bacillus, 
Micrococcus catarrhalis, staphylococci and streptococci. The 
former two also invade the blood stream. After:5 to 24 hours the 
mouse is killed and the peritoneal fluid and heart’s blood streaked 
on blood agar plates. Gram stain and Hiss capsule stain of the 
peritoneal exudate are examined. Then the peritoneal fluid 
is washed out with 5 c.c. of salt solution and the suspension is 
transferred to a centrifuge tube, whirled at low speed to throw 

"Blake, F. G.: Journ. Exp. Med., 1917, 26, p. 67. 


270 SPECIFIC MICRO-ORGANISMS 


down the leukocytes and fibrin. The supernatant fluid is then 
transferred to a clean tube and whirled at high speed to sediment 
the bacteria. The supernatant fluid is discarded and the bacter- 
ial sediment is suspended in salt solution sufficient to make a 
translucent mixture. Five tubes’ are then set up as follows: 

Tube 1—o.s§ c.c. of 1 : 20 dilution Type I serum, 0.5 c.c. of 
the bacterial suspension. 

Tube 2—0o.5 c.c. of undiluted Type II serum, 0.5 c.c. of the 
bacterial suspension. 

Tube 3—0.5 c.c. of 1: 20 dilution Type II serum, 0.5 c.c. 
of the bacterial suspension. ; 

Tube 4—0.5 cc. of r: 5 dilution of Type III serum, 0.5 cc. 
of the bacterial suspension. 

Tube s5—o.1 c.c. sterile ox bile, 0.3 c.c. of the bacterial 
suspension. 

The tubes are immersed in water bath ‘at 37° C. for one hour. 
Agglutination in the homologous type serum is usually prompt. 
If no agglutination occurs after one hour and the organism is 
a bile soluble encapsulated diplococcus, it is classed as a pneu- 
mococcus of Group IV. If agglutination is present in Tube 2 
but absent in Tube 3, the organism is classed’ in Subgroup II. 

Various simpler methods! of typing pneumococci have been 
devised, but they are less reliable even in expert hands. 

Kyes® has injected chickens with massive doses of pneumo- 
cocci and has obtained a powerful anti-serum which seems to be 
potent against various strains and types of pneumococci. Fur- 
ther confirmation of his results should be awaited. 

Streptococcus Viridans.—Schottmueller® has found a strepto- 
coccus, resembling in some respects the pneumococcus, in the 
blood of cases of subacute endocarditis or endocarditis lenta. 
On the blood-agar plates the colonies appear after two to five 


* Mitchell and Muns; Journ, Med. Rsch., 1917, 37) P. 339- 
Avery, O. T.; Journ. Amer. Med. Assn., 1918, 70, p. 17. 
2 Kyes, P. Pott Amer. Med. Assn., 1911, 56, p. 1878; Journ. Med. Rsch., 


1918, 38, DP. 495. 
3 Muenchener med. Wochenschr., 1903 (I), No. 20, p. 849. 


COCCACEZ AND THEIR PARASITIC RELATIONSHIPS 271 


days as opaque granules surrounded by a cloudy but distinctly 
greenish zone. The organism is being found very frequently 
in cases of subacute endocarditis,! and is apparently the specific » 
cause of this particular fairly well-defined type of endocarditis. 
The same organism is found normally in the mouth and pharynx 
and has been designated also ds streptococcus salivarius. It is 
also commonly present in abscesses at the roots of the teeth. 

Streptococcus Pyogenes.—Bacteria were observed in pyemic 
abscesses by Rindfleisch in 1866 and in the following years this 
observation was confirmed by numerous pathologists. Klebs 
(1870-71) recognized the ‘‘ Microstoron septicum”’ as the cause of 
wound infections and the accompanying fever, as well as the 
resulting pyemia and septicemia. Ogston (1882) first clearly 
distinguished between -the chain-form, streptococcus, and the 
grape-form, staphylococcus, of the pus cocci, not only on the 
basis of their grouping but also in respect to the types of inflamma- 
tion with which they are associated. Pure cultures were first 
obtained by Fehleisen (1883) from erysipelas (Streptococcus 
erysipelatos) and by Rosenbach (1884) from the pus of wounds 
(Streptococcus pyogenes). The former produced typical erysipe- 
las by inoculating the human skin with his cultures. There 
is no specific distinction between the streptococci found in ery- 
sipelas and those found in other lesions. The difference in the 
pathological process depends rather upon the portal of entry of 
the infection, the virulence of the microbe and the resistance of 
the host. 

Streptococcus pyogenes lives naturally upon the mucous mem- 
branes, especially in the pharynx, nose and mouth, the intestine 
and on the vaginal mucosa. Such streptococci found in normal 
individuals are relatively non-virulent. Virulent streptococci 
occur in erysipelatous lesions of the skin, in infected wounds 
on the inflamed pharyngeal mucosa, and in the lochia, uterine 
wall and in the circulating blood in puerperal fever. Streptococci 
are frequently found in pyemic abscesses, bacteremia, meningitis 

' Major, Johns Hopkins Hosp. Bull., 1912, Vol. XXIII, pp. 326-332. 


, 


272 SPECIFIC MICRO-ORGANISMS 


and pneumonia. It seems probable that these virulent races 
originate from the ordinary relatively harmless parasitic forms in 
some instances, when an opportunity is presented for successful 
invasion of tissues by a lowered resistance of the host, and that 
by successive transfer from one susceptible individual to another 
the virulence is still further enhanced. 

The individual cells of a chain vary in size from 0.6 to 1.54 
and in form from flattened disks to long ovals. The chains are 
variable in length and in general the more virulent types form 
longer chains in broth cultures. In old cultures the cells are very 
-irregular in size, and it was once supposed that the larger spheres 
were special resistant forms, “arthrospores.”’ They ‘are now 
regarded as involution or disintegrating forms. The streptococcus, 
stains readily and is Gram-positive. 

Cultures on ordinary media are relatively poorly developed 
and of short life. Broth or glucose broth serves very well, and 
a few cultures in series may be obtained on glycerin agar or glu- 
cose agar. Léffler’s blood serum is better than these. Serum 
agar, ascitic-fluid agar and blood agar are the best solid media 
and ‘ascitic-fluid broth is an excellent fluid medium for cultiva- 
tion of streptococci. Blood agar is especially valuable in plating 
pus or exudates because of the rather characteristic appearance 
of the small colony surrounded by a very clear zone of hemolysis ~ 
which the streptococcus produces on this medium. In making 
cultures from the blood in bactéremia, plain agar previously 
melted and cooled to 45° C. is mixed with freshly drawn blood 
of the patient and allowed to solidy in a Petri dish. In other 
cases naturally sterile defibrinated rabbit’s blood may be used, 
the technic of plating being analogous to that described for the 
gonococcus. The streptococcus grows very slowly below 20°C. 
and poorly. in ordinary gelatin, which it does not liquefy. On 
solid media, agar or serum-agar, at 37° C., small round elevated 
colonies develop, 0.5 to 1.0 mm. in diameter, and they tend to 
remain discrete. In broth only a slight cloud develops, but 
considerable granular deposit made up of streptococci is found at 


COCCACEZ AND THEIR PARASITIC RELATIONSHIPS 273 


the bottom of the tube. Various carbohydrates are fermented 
with the production of acid and without formation of gas, but 
the behavior of streptococci toward these substances seems so 
variable that the attempts to utilize the fermentative power as 
a basis for classifying the streptococci has not led to wholly satis- 
factory results. The differences in fermentative power seem to 
depend more upon vigor of growth than upon essential qualita- 
tive differences between the streptococci tested.! 

The streptococcus is relatively very resistant to heat, at times 
requiring one to two hours heating at 65° C. or one hour at 70° 
C. in order to insure sterility, according to V. Lingelsheim. Most 
investigators have found 60° C. for twenty minutes sufficient. 
Its poisons seem to be chiefly intracellular and set free upon dis- 
integration of the organisms. Soluble poisons have nevertheless 
been found in some cultures. 

| Laboratory animals are not very susceptible to inoculation 
with streptococci: White mice and rabbits are most useful, 
and they ordinarily succumb to intraperitoneal injection of. 
virulent strains. 

The enormous importance of the streptococcus as a cause 
of sickness and death before the aseptic era is difficult to realize 
at the present time. Veritable epidemics of streptococcus in- 
fection in the surgical and obstetrical wards of hospitals made 
this one of the most dreaded of diseases. Even to-day the 
virulent streptococcus is held in great respect by many surgeons, 
and cases of erysipelas and other recognizable active streptococcus 
infections are commonly excluded from surgical wards. 

In war wounds the streptococcus is a common and serious 
infectious agent. Its presence may be rapidly detected by in- 
oculating the wound exudate into broth containing a bit of liver 
or other tissue. 

Erysipelas is an acute febrile disease diesel by a local 
redness and edema of the skin which tends to spread to contigu- 
ous areas. In the lymph spaces beneath the epithelium there is a 


1 V, Lingelsheim in Kolle und Wassermann, Handbuch, 1912, Bd. IV, S. 462. 
18 


274 SPECIFIC MICRO-ORGANISMS 


collection of leukocytes and serum, and the streptococci are also 
found here, especially at the periphery of the reddened area. 
In follicular tonsillitis and many cases of pseudo-membranous 
angina as well as in the pharyngitis of scarlet fever, streptococci 
occur in large numbers, and doubtless bear a causal relation to 
at least a part of the pathological process. In true diphtheria, 
streptococci seem to play rather frequently the réle of important 
secondary invaders. From the pharynx the streptococcus 
may gain access to the middle ear and the mastoid cells, to the 
meninges, to the trachea, bronchi and lungs, setting up purulent 
inflammations in any of these locations. It is an important 
secondary invader in pulmonary tuberculosis. The streptococcus 
seems also to cause enteritis, particularly in infants. In the puer- 
perium, streptococci are practically always present in the lochia. 
In spite of many attempts to differentiate between virulent 
and non-virulent types in this situation, it is still impossible to 
distinguish them. Probably local conditions in the uterus as 
well as the general condition of the patient have much to do in 
determining her resistance to infection of the uterine wall with 
these normal streptococci. Undoubtedly the frightful epidemics 
of puerperal fever in some hospitals previous to 1875 were due to 
the transference of virulent organisms from patient to patient 
by the attending physicians and nurses. This was first suggested 
by Holmes (1843) and more definitely proven by Semmelweiss 
(1861), but their ideas received little credence until the last quar- 
ter of the nineteenth century. Streptococcus bacteremia is 
commonly a terminal phenomenon, but it may occur without 
immediate fatal issue, and may result in endocarditis and strepto- 
coccus arthritis. 

_ Immunity to streptococcus infection is slight in degree and 
very temporary. Koch showed that erysipelas could be repeat- 
edly produced on the same area of the skin by inoculation at inter- 


vals of 10 to 12 days. Rabbits and horses acquire a high degree 


of immunity when treated with gradually increasing doses of 
many different strains of streptococci. The serum-of such 


\ 


COCCACEZ AND THEIR PARASITIC RELATIONSHIPS 275 


animals has a marked protective influence when injected into 
animals and has been employed in treating human infections, 
in some cases with success, while in others the serum has appar- 
ently exerted no influence on the course of the disease. In local- 
ized chronic streptococcus infections, treatment with autogenous 
bacterial vaccines (bacteria suspended in salt solution and killed 
by heat) seems to produce favorable effects in some cases. 

Streptococcus Lacticus (Micrococcus Ovalis).—This is a 
variety of streptococcus growing normally in the intestine and 
of special importance as the cause of the normal souring of milk. 

The classification of the streptococci has been studied exten- 
sively in recent years. The fermentative effects upon blood and 
upon various sugars, especially lactose and mannite, are important 
criteria. The advanced student should consult the article of 
Blake! and the monograph of Brown,’ both of which give refer- 
ences to the literature. 

Staphylococcus (Micrococcus) Aureus.—-By the early ob- 
servers (Rindfleisch, Klebs) this organism was not distinguished 
from the streptococcus. Pasteur in 1880 obtained it in broth 
cultures from pus. Ogston in 1882 clearly distinguished it from 
the streptococcus. Rosenbach (1884) by his extensive inves- 
tigations established the position of the staphylococcus as a 
cause of wound infection and of osteomyelitis. 

Staphylococci have their natural habitat on the skin, in the 
mouth, in the nasal cavities and in the intestine, without the 
presence of inflammation. More virulent forms occur in in- 
fected wounds, furuncles, carbuncles, various localized purulent 
inflammations, bacteremia (staphylococcemia), endocarditis, 
osteomyelitis, meningitis and pneumonia. 

The cell is spherical, 0.7 to o.gu in diameter. Division takes 
place in various planes, giving rise to irregular bunches of cocci. 
The organism stains readily and is Gram-positive. Cultures 


1 Blake, F. G., Journ. Med. Rsch., 1917, 36, p. 99. 
2 Brown, J. H., Monographs of the Rockefeller Institute for Medical Research, 
No. 9, 1918. 


276 SPECIFIC MICRO-ORGANISMS 


are readily obtained on all the common media and growth occurs 
between 9° and 42°, best at 37° C. Broth is diffusely clouded 
with abundant sediment. In gelatin stab-culture, growth occurs 
all along the line of inoculation with funnel-shaped liquefaction 
(Figure 109). On agar slant the growth is con- 
fluent and yellowish after 24 hours. There is 
similar growth on Léffler’s serum, often with 
liquefaction of the medium. 

The staphylococcus is relatively resistant to 
heat and chemical germicides. It is killed at 
62° C. in ten minutes and at 70° C. in five 
minutes. V. Lingelsheim’ found it more re- 
sistant, requiring ten minutes at 80° C. and 
an hour at 70° C. to kill his strains, but his 
figures cannot be accepted without further con- 
firmation.? It is about as resistant to chemical 
poisons as any of the sporeless bacteria, and 
is commonly employed as a test object in the 
investigation of germicides. Mercuric chloride 
I-1000 requires three to five hours to kill staph- 
ylococcus cultures and much longer if the 
organisms are present in pus. Carbolic acid, 
3 per cent, kills them in two to ten minutes. 

Fic. =a The pigment is a lipochrome and is pro- 
culture Staphylococcus duced only in the presence of oxygen. The 
iii ac iat tryptic ferment diffuses out of the cells and is 
capable of liquefying gelatin, albumen and fibrin. The staphy- 
lococcus produces a soluble poison which kills leukocytes 
(leukocidin) and others which dissolve red blood cells (staphy- 
lolysin) and cause clumping of red blood cells (agglomerin). 
These substances are true soluble toxins and they are destroyed 
by heating to 80° C. Other soluble poisons seem also to be pre- 
sent. -The bacterial cells killed by heat are only slightly toxic, 


1 Neisser: Kolle und Wassermann, Handbuch, 1912, Bd. IV, S. 361. 
? Compare with similar tests on streptococci by v. Lingelsheim, p. 273. 


COCCACEZ AND THEIR PARASITIC RELATIONSHIPS 277 


yet it is very probable that in the disintegration of the cocci in 
an inflammatory process more poisonous substances may be 
derived from their cell protein. 

Rabbits are the animals of choice for inoculation with staphy-- 
lococci. Intravenous injection with virulent cultures usually. 
causes multiple abscesses in the internal organs with death. in 
4 to 8 days. Typical endocarditis has been produced by injected 
organisms from potato cultures, and with greater certainty when 
the heart valves are injured mechanically, especially in young 
rabbits. Osteomyelitis sometimes follows intravenous injection 
in growing rabbits, especially if the bone be slightly injured 
at the time of inoculation. In man, typical furuncles and carbun- 
cles have been produced by rubbing pure cultures on the skin 

‘ (Garré, 1885) and by subcutaneous injection. 

In man this organism is a frequent cause of local purulent 
inflammations, and it Sometimes gives rise to pyemic abscesses 
and general bacteremia. Recurrent furuncles and carbuncles 
are ordinarily due to staphylococci. 

Animals have been immunized to staphylococci but the serum 
obtained from them has relatively slight value in treatment. 
Specific treatment by means of dead bacterial cells, bacterial 
vaccines, has been developed by A. E. Wright and has proved 
-its value in the treatment of chronic furunculosis. A suspension 
in salt solution of bacterial cells from an agar slant, sterilized 
by heating to 60-65° C. for 30 minutes and standardized by 
microscopic count of the bacterial cells, is employed. Doses 
from 50 millicn to 1000 million bacterial cells are injected two 
or three times a week for a long period of time, the size and fre- 
quency of dosage being governed by the clinical condition of the 
patient. Determination of the opsonic index is probably un- 
necessary and is now quite generally neglected. Autogenous 
vaccines (made with the staphylococcus isolated from the patient) 
are usually superior to stock vaccines. 

Staphylococcus Albus.—This is quite similar to Staphylococcus 
aureus in all respects except pigment production. Usually, 


278 SPECIFIC MICRO-ORGANISMS 


but not always it is less virulent. Staph. epidermidis (Welch) is 
an avirulent variety of Staph. albus, very abundant on the normal 
skin. Many other varieties of staphylococci have been described. 

Micrococcus Tetragenus.—This organism occurs in lung cav- 
ities in phthisis, and in the sputum, usually in groups of four 
cells, tetrads, enclosed in a transparent capsule. It is Gram- 
positive, grows on ordinary media and does not liquefy gelatin. 
White mice and guinea-pigs are susceptible and ordinarily die 
of general bacteremia in two to six days after inoculation. The 
pathogenic réle of the organism in man is doubtful. 

Sarcina Ventriculi—Goodsir in 1842 observed sarcines’ in 
vomitus. The coccus is large, 2.54 in diameter, and occurs in 
cubes of eight cells or as large conglomerates of these. It grows 
on ordinary media, usually producing a yellow pigment. It 
is found in the stomach in some conditions in which the acidity 
of the gastric juice is diminished. It is apparently non-patho- 
genic. 

Sarcina Aurantiaca.—This is a common saprophytic coccus 
found in fermenting liquids and occasionally in the air. It 
grows well on ordinary media and liquefies gelatin. An orange 
pigment is produced. Typical packets are produced in liquid 
media, especially in hay infusions. 

Micrococcus (Planococcus) Agilis—This oganism occurs in 
surface waters. It liquefies gelatin and produces a rose-red 
pigment on agar and potato. Its remarkable feature is the pos- 
session of a flagellum and active motility. It is Gram-positive. 


CHAPTER XVII 
BACILLACEZ: THE SPOROGENIC AEROBES 


The aérobic spore-forming bacilli are essentially inhabitants 
of the soil and the fermenting organic material likely to occur 
there. Along with a few species of this group we shall consider 
one pathogenic sporogenous bacterium, the anthrax bacillus, 
which resembles them very closely except in its virulence for 
animals and its lack of active motion, both of which may perhaps 
justly be regarded as variations from the group type due to its 
parasitic mode of life. 

, Bacillus Mycoides.—This organism is universally distributed 
in fertile soils and also occurs in surface waters and in the air. 
It is a large rod with rounded ends, usually growing in threads. 
Large median spores are formed without distorting the cell. 
It is motile but rather sluggish. Growth occurs on all ordinary 
media. In gelatin stab-culture, thread-like processes extend 
out on alj sides from the line of puncture giving the appearance 
of an inverted pine tree. Later the gelatin becomes entirely 
liquefied. The organism is an important agent in the decompo- 
sition of plant residues in the soil. It is without pathogenic 
properties. 

Bacillus (Mesentericus) Vulgatus.—This is another widely 
distributed soil bacterium. It is commonly called the potato 
bacillus. The cell is short and relatively thick with rounded 
ends, actively motile, often in pairs or threads. Large spherical 
median spores are produced without distortion of the cell. These 
spores are very resistant to heat and germicides, sometimes 
surviving the temperature of boiling water for several hours. 
B. vulgatus grows well on all ordinary media. Gelatin is liquefied. 
Milk is coagulated and then digested. On potato a wrinkled 

279 


280 SPECIFIC MICRO-ORGANISMS 


membrane is produced, so characteristic that the name ‘“‘mesen- 
tericus’”’ was applied to this species. It is not pathogenic. 
Bacillus Subtilis—Bacillus subtilis, or the hay bacillus, is 
abundant in the soil and on the surface of plants, and common 
in surface waters and in the air. It is readily obtained by boiling 
hay in water and then setting the infusion aside for a few days. 
The cell is relatively large, about 1.24 wide by 5x long, with ends 
somewhat rounded. Long threads are commonly formed. It 
is motile with peritrichous flagella. Large oval median spores 


Fic. 110.—Bacillus subtilis. Xz1o000. 


are formed without distortion of the cell and these are almost as 
resistant as the spores of the potato bacillus. B. subtilis grows 
rapidly on ordinary media in the presence of air, best at about 
30° C. Gelatin is liquefied and milk is digested. The organism 
is typically saprophytic, but it has been found growing in the 
intestine by some investigators, and has been found in a few in- 
stances in nections of the human eye, cases of panophthalmitis 
following injury.! 


: Silberschmidt, Annales de l'Institut Pasteur, 1903, Vol. XVII, pp. 268-287; 
Also see Kneass and Sailer, Univ. Penn. Med. Bull., June, 1903, Vol. XVI, pp. 
I3I-133. ; 


BACILLACEZ! THE SPOROGENIC AiROBES 281 


‘Bacillus (Bacterium) Anthracis.—Pollender in 1849 and 
_Davaine and Rayer in 1850. observed thread-like bodies in the 
blood of animals dying of anthrax. Robert Koch in 1876 ob- 
tained pure cultures of the organism, using the aqueous humor of » 
the ox’s eye as culture medium. He saw the small rod-shaped 
bodies found in the anthrax blood elongate into threads in this 
- medium, and observed the formation of the bright refractive 
bodies in these threads, which he correctly recognized as spores. . 
Finally by inoculating healthy animals with his cultures he pro- . 


Fic. 111.—Anthrax bacilli in the capillaries of the liver of a mouse. 


duced typical anthrax in them, thus proving conclusively for the 
first time the causal relation of a bacterium to a disease. 

The anthrax bacillus occurs in the blood and throughout the 
tissues of animals suffering from anthrax, and in the excretions 
of such animals. Its spores occur on hides and in wool derived 
from‘ anthrax animals. Furthermore, the soil of fields where 
anthrax animals have grazed harbors these organisms for many 
years. It seems probable that the bacilli multiply in the soil 
during the warm wet seasons and it is certain that the spores 
may lie dormant for as long as ten years in dry places. 


282 SPECIFIC MICRO-ORGANISMS 


The cell is about 1.254 wide and 3 to 10 long, with rounded 
ends when single, but in the threads the contiguous ends are 


Fic. 112.—Bact. anthracis. Spore production. (From Marshall after Migula.) 


-square-cut. In the circulating blood the bacilli are single or in 
pairs and spores are never formed in the animal body (Fig. 111). 
In cultures long threads are produced and spores are usually 


Fic. 113.—Bact. anthracis. Colony upon’a gelatin plate. xr100. (After Fraenkel 
: ,, and Pfeiffer.) 


formed after 24 to 48 hours (Fig. 112). The anthrax bacillus 
is aérobic and grows readily on all ordinary media, best at 37° C. 


BACILLACEZ: THE SPOROGENIC ALROBES 283 


Gelatin is slowly liquefied. The colony presents a very char- 
acteristic appearance, especially as it grows on gelatin, which is 
due to the large coils of long parallel threads, of which the colony 
is composed. The vegetative bacillus is rather easily killed but 
the spores may survive boiling in water for 5 minutes and in some 
instances as long as half an hour when afforded some mechanical. 
protection. Chemical germicides cannot be relied upon to destroy 
the spores. Sterilization in the autoclave is the safest method 
of disposing of anthrax material. 

Anthrax is a disease which occurs spontaneously in cattle and 


Fic. 114.—Bact. anthracis. Showing the thread formation of colony. (After Kolle 
- and Wassermann.) 


sheep and-rarely in horses, swine and in man. The disease is 
produced by inoculation in many other animals. Mice, guinea- 
pigs and rabbits are susceptible in the order named. The disease 
is common in European and Asiatic stock-raising districts and 
in Argentine Republic. Several local epizodtics have occurred in 
the United States and a few cases of human anthrax. Experi- 
mental anthrax is readily produced in susceptible animals by 
subcutaneous inoculation, less certainly by feeding the spores. 
In the acute form the bacilli are found in large numbers every- 
where in the blood, and this is the common picture in cattle, 
sheep, rabbits, guinea-pigs and mice. Chronic forms occur, 


284 SPECIFIC MICRO-ORGANISMS 


however, either because of lowered virulence of the germ or ‘of 

increased resistance of the host, and in these cases the bacteria. 
may be very scarce and difficult to find microscopically, even 

after death of the animal. Cultures from the spleen will usually 

show the presence of the bacillus there. The mechanism by 

which the bacillus causes death is unknown. In the acute cases, 

as in the mouse, the bacilli are so abundant in the blood that 

mechanical interference with the circulation seems a plausible 

explanation, but this certainly does not suffice for other types of 

the diseasein which chemical poiscning must play the chief 

réle. So far it has not been possible to demonstrate any powerful » 
poisons in cultures of the anthrax bacillus. It is probable that 

the essential poisons are produced by a reaction between the 

substance of the bacillus and the fluids of the host, particularly 

the enzymes of the latter, which cause disintegration of the bac- 

terial bodies. 

The infection is acquired by grazing animals through the 
alimentary tract primarily, but also to some extent by inoculation 
(contact, flies, intermediate objects). In man there are three 
recognized types (a) malignant pustule, (6) pulmonary anthrax, 
and (c) intestinal anthrax. Malignant pustule results from in- 
oculation of the skin, especially in those who handle hides or care 
for anthrax animals. It is at first a local pustular and necrotic 
lesion tending to involve contigious tissue by extension, but soon 
invading the lymph vessels and walls of the veins. The bacteria 
thus gain the blood stream and a rapidly fatal general bacteremia 
supervenes. Recovery sometimes occurs before the disease be- 
comes generalized. Several instances of malignant pustule of 
the face were observed in soldiers in 1918, the infection being 
derived from shaving brushes, in some of which authrax bacilli 
were found. Pulmonary anthrax is caused by inhalation of an- 
thrax spores (woolsorter’s disease). Intestinal anthrax is un- 
common in man but has occurred. Both are very fatal forms of 
the disease. 

Immunity to anthrax was first successfully produced by Pas- 


BACILLACEZ: THE SPOROGENIC AEROBES 285 


teur through vaccination with attenuated living cultures. Broth . 
cultures inoculated with bacilli taken directly from the animal 
body were grown at 42° C. to 43° C. At this temperture spores 
are not produced and the bacillus gradually loses its virulence. 
When it will no longer kill guinea-pigs but will still kill mice the 
strain is again grown at 37° C. and injected into cattle and sheep 
as the first vaccine. Twelve days later a second vaccine is in- 
jected, which is a somewhat more virulent culture, still capable 
of killing guinea-pigs but not powerful enough to cause fatal in- 
fection of rabbits. As a result of these two treatments, nearly 
-all animals become immune to the natural disease or to inocula- 
tion with fully virulent cultures. Sobernheim! and Sclavo? have 
induced a high degree of immunity in sheep and in asses by re- 
peated injection of the bacilli, and have found the serum of such 
hyper-immune animals to be protective and curative upon in- 
jection into other animals. The injection of this serum along 
with a dose of living culture of about the strength of Pasteur’s 
second vaccine has been employed in immunizing cattle and 
sheep. All the necessary treatment is thus given at one time. 
The serum has also been successfully employed in conjunction 
with the appropriate medical and surgical measures in the treat- 
ment of malignant pustule in man.’ 

1 Sobernheim: Zeitsch. f. Hyg., 1897, XXV, pp. 301-356; Centralbl. f. Bakt., 
1899, XXV, p. 840. 

1 Sclavo: Centralbl. f. Bakt., 1899, X XVI, p. 425. 

3 For a discussion of treatment of human anthrax consult Boidin, Vignaud and 


Fortineau, Presse Médicale, Aug. 14, 1912; also Becker, Munch. med. Wochenschr., 
Jan. 23, 1912. ' 


CHAPTER XVIII 
BACILLACEZ: THE SPOROGENIC ANAEROBES 


The bacteria of this group are hindered in their development 
by the presence of free oxygen and their artificial culture is ordi- 
narily successful only when they are protected from oxygen, at 
least in the early stages of development. Like the sporogenic 
aérobes, they live in the soil, but they are associated here more 
especially with decomposing materials of animal origin, and are 
less frequently found in soils which have not received fertilizers 
from animal sources. There is good reason to believe that their 
essential habitat is the intestinal canal of animals, especially the 
mammals, and that their life in the soil does not represent the 
most active stage of their existence, but that they reach the soil 
with animal excreta and the bodies of dead animals and continue 
to live in the soil for a considerable period. For this group of 
bacteria the Committee of the Society of American Bacteriologists 
has suggested the generic name, Clostridium Prazmowski 1880. 

Clostridium Edematis (Vibrion septique).—Pasteur in 1877 
injected infusions of putrid flesh into laboratory animals and 
produced a fatal subcutaneous edema with penetration of the 
bacteria into the blood in some instances. The organism which he 
called ‘“Vibrion septique”’ was found to be an obligate anaérobe, 
the first anaérobic organism ever recognized. Koch (1881) 
studied the organism in pure culture on solid media and named 
it Bacillus edematis maligni. 

The bacillus is very widely distributed in soil and dust, and 
is very common in the feces of herbivorous animals. It is es- 
pecially abundant in putrefying animal matter. The cell is about 
tm thick by 3 in length, although considerable variation in size 


and shape occurs. It is usually slightly motile and possesses 
286 


’ 


BACILLACEZ: THE SPOROGENIC ANAEROBES 287 


peritrichous flagella, stains readily, is only relatively Gram-posi- 
tive, some of the cells being decolorized by prolonged treatment 
with alcohol. The spores are central, or intermediate in position, 
with bulging of the cell. 

‘In cultures Cl. edematis is a strict anaérobe. It liquefies gela- 
tin. Milk is slowly coagulated and the coagulum digested, the 
reaction remaining alkaline to litmus. The cultures have a foul 
odor. The spores withstand boiling sometimes for 2 to 3 hours. 
The morphological and physiological properties of this organism 
are quite variable and the many intermediate types between it 
and B. feseri makes distinction between the two species somewhat 
difficult. 

In animals and man, malignant edema occurs spontaneously 
as a wound infection, but it is not very common. It has been 
observed most frequently in horses and in new-born calves. The 
guinea-pig is susceptible. In general a mere injection of the 
bacilli fails to produce serious disease. The presence of foreign 
bodies or extensive tissue destruction favors the infection. 

In war wounds the Vibrion septique is an important cause of 
gaseous edema, usually in association with other anaérobic 
bacteria. 

Clostridium Feseri.—Feser and Bollinger (1875-1878) observed 
the large narrow rods in the diseased tissues and exudates of 
symptomatic anthrax or black leg, a fatal disease of cattle and 
sheep. Man is not affected. Arloing, Cornevin and Thomas 
(1884) obtained the organism in culture. The organism is a 
strict anaérobe and resembles B. edematis very closely. Black 
leg is a local emphysematous inflammation usually beginning in 
one leg of cattle or sheep, rapidly extending and resulting in death 
as arule. Immunity is obtained by injecting small doses of the 
virulent bacteria or by injecting attenuated organisms, and also 
by injecting the virus together with an immune serum." 

Clostridium Perfringens (Bacillus Welchii)—Welch and 
Nuttall in 1892 discovered this organism at autopsy in a body 

1 Kitt, Kolle and Wassermann, Handbuch, 1912, Bd. IV, S. 819-836. 


288 SPECIFIC MICRO-ORGANISMS 


Fic. 115.—Cl. perfringens in agar 
culture, showing gas formation: 


showing general emphysema of the 
tissues and gas bubbles in the blood- 


vessels. They obtained cultures by 


anaérobic methods and caused similar 
post-mortem emphysema in the bodies 
of rabbits. The organism lives and 
multiplies in the intestine of man and 
other-‘mammals, is widely distributed 
in the soil and is commonly present 
in milk and other animal food prod- 
ucts. The cell is a large rod sur- 
rounded by a capsule when grown 
on media rich in protein or in the 
animal body. The width of the cell 
(without capsule) varies! from 1.1 
to 1.74 with a mean of 1.34 and the 
length from 2.6 to 7.64, with an 
average of 4.6u, the measurements 
being made on organisms grown in 


_-an agar stab-culture 24 hours at 


37° C. When grown in blood broth 
the germ is capsulated and_ the 
measurements, including the capsule 
are as follows: width 1.9 to 2:54 with 
average of 2.1% and length, 2.8 to 
6.6% with average of 4.74. Usually 
the organism is non-motile, but flag- — 
ella can sometimes be demonstrated. - 
In the intestine and-in protein media 
the organism forms spores, usually 
median without bulging of the cell, 
but these are not commonly observed 


1 The measurements are taken from Kerr, The Bacillus welchit, Thesis, Univ. 


of Illinois, 1909. 


BACILLACEZ: THE SPOROGENIC ANAHROBES 289 


in cultures. The organism is a strict anagrobe. Its most striking 
property is the enormously rapid production of gas in media con- 
taining dextrose or lactose. Cultures are obtained most readily 
by heating a suspension of feces to 80° C. for 15 minutes and in- 
oculating it into glucose broth mixed with blood in a Smith fermen- 
tation tube. After 24 to 48 hours incubation its presence will 
usually be revealed by abundant production of gas. Milk is 
coagulated and rendered acid with an abundant production of gas 
(stormy fermentation). On blood-agar plates incubated in 
hydrogen, the colony is round with regular outline and surrounded 
by a clear zone of hemolysis. . 

Emphysematous gangrene occurs in man as a rapidly extend- 
ing, very fatal disease, due to the infection of wounds with this 
organism. The presence of necrotic tissue seems to be necessary 
in order that the organism may gain a foothold, but: when once 
begun the inflammation may extend with great rapidity. The 
gas found in bodies at autopsy is usually the result of an agonal 
or a post-mortem invasion by the bacilli from the intestine. 

There are several other types of sporogenic anaérobes of the 
same general nature as Cl. edematis, Cl. feseri, and Cl. welchii, 
which live normally in fertilized soil and in the intestines of 
animals. The organisms of .this group have assumed great 
importance in modern warfare as the causes of anaérobic wound 
infection, variously termed gaseous gangrene, gaseous edema or 
toxic edema. ‘The development of the disease depends to some 
extent upon the presence of foreign bodies or devitalized or partly 
disorganized tissue in a wound. It may appear early and run a 
rapid course to death. Such cases are usually infected with Ci. 
perfringens. Late, more slowly progressing gaseous: edema is 
often due to a mixture of bacteria, including the Vibrion septique 
or Cl. edematiens of Weinberg and Seguin or both of these. The 
monograph! of these authors should be consulted by those students 
who are interested in the anaérobic infection of wounds. These 
authors have made substantial progress in the production of 


" Weinberg et Seguin: La gangrene gaseuse, Masson et Cie, Paris, 1918. 
19 


290 SPECIFIC MICRO-ORGANISMS 


anti-sera for the prophylaxis and treatment of these wound’ 
infections. 

Clostridium Tetani—Tetanus has been recognized as a com- 
plication of wounds since the time of Hippocrates. Forscher, 
Carle and Rattone, in 1884, first proved it to be inoculable by in- 
jecting pus from a human case into 12 rabbits, of which 11 died 
of tetanus. Nicolaier in 1884 produced tetanus by injecting soil 
into mice, guinea-pigs and rabbits, and found a slender bacillus 
in the animals at the point of inoculation. He was able to prop- 
agate the bacillus in mixed culture on coagulated sheep’s serum. 
Kitasato obtained the first pure cultures by subjecting the mixed 
culture to a temperature of 80° C. for an hour, inoculating agar 
plates and incubating them in an atmosphere of hydrogen. With 
his pure cultures, he caused typical tetanus in animals. — 

The organism occurs in the soil which has received animal 
fertilizers and in the intestine of herbivorous mammals. The 
bacterial cell is 0.3 to 0.54 wide and 2 to 4 long, single in young 
cultures, but often joined end to end to form long threads in older 
cultures. Jt is motile and possesses abundant peritrichous’ 
flagella. The spore is very characteristic. 1t is usually spherical, 
1 to 1.54 in diameter, situated at the extremity of the cell, giving 
it the appearance of a’drumstick. The bacillus stains readily 
and is Gram-positive. , 

Isolation of Cl. tetani from mixed material or from wenn 
known to contain it is not always easy. The niaterial should 
be planted in glucose broth and incubated in hydrogen at 37° C. 
for 2 to 3 days. Microscopic examination of the sediment may 
then reveal the drumsticks. Kitasato’s procedure should then be 
followed, employing agar distinctly alkaline to litmus and con- 
taining 2 per cent of glucose. If many other spore-forming bac- 
teria are present in the mixture, special procedures, are necessary, 
such as preliminary culture for 8 days at 37° C. ina deep stab in 
coagulated rabbit’s blood with subsequent heating to 80° C. to 
get rid of Cl. edematis, or culture for 8 days at 37° C. in milk 
with subsequent heating to get rid of Cl. perfringens. Aérobic 


BACILLACEE: THE SPOROGENIC ANAEROBES 2g1 


spore-formers may be eliminated by successive transfers in 
animals. 

The spores of Cl. tetani resist the temperature of boiling water 
for 5 to 30 minutes. Biological products to be introduced into 
the human body need to be sterilized in the autoclave or else 
carefully examined by anaérobic culture methods to insure their 
freedom from tetanus spores. The danger of infection from this 
source has been emphasized by Smith.! 

The colony in glucose gelatin or glucose agar consists of a 
compact center with slender, radiating, straight or irregularly 
curved threads about the periphery. Liquefaction of* gelatin 
‘becomes evident in stab-culture after about two weeks at 20° C. 
Milk is sometimes but not always coagulated and the casein is 
eventually digested. 

The cultures of the tetanus bacillus are extremely poisonous, 
especially so when they are developed under very strict anaérobic 
conditions. A nerve poison, tetanospasmin, and a hemolytic 
poison, tetanolysin, are present. The former is the more impor- 
tant constituent of the tetanus toxin. Neutral or slightly alka- 
line plain nutrient broth, incubated in an atmosphere of hydrogen 
for ten days after inoculation gives the most powerful toxin. 
The bacteria-free fluid from such a culture has been found to kill 
a mouse of 10-grams weight in a dose of 0.000 005 c.c. The toxin 
is unstable in solution but very stable when dried. Dry material 
of which 0.000 000 1 gram is the fatal dose for a mouse is readily 
obtained. The watery solution loses it toxicity when heated to 
60° C. for 20 minutes, but when dry the toxin withstands heat- 
ing at 120° C. for an hour. 

Tetanus presents essentially the same picture in inoculated 
animals as in the natural disease, which is indeed, as a general 
rule, merely an accidental inoculation. The presence of insoluble 
material and of other bacteria mixed with them in a wound favors 
the development of tetanus bacilli. The tetanus bacilli always 
remain localized near the point of inoculation and may be hard 


1 Journ. A. M..A., Mar. 21, 1908, Vol. L., pp. 929-934. 


292 SPECIFIC MICRO-ORGANISMS 


to find. The poison produced by the organisms is probably ab- 
sorbed by the nerve endings! and transmitted to the central nervy- 
ous system through the axis cylinders or in the perineural lymph . 
spaces of the motor neurones rather than through the blood 
stream. The symptoms arise after the poison reaches the central 
nervous system in sufficient concentration to stimulate the nerve 
cells. In guinea-pigs and mice the spasm always begins near the 
point of inoculation, but in man and the large mammals it often 
begins in the muscles of the jaw and neck regardless of the location 
of the wound. Wassermann and Takaki have shown that o.1 


Fic. 116.—Tetanus bacilli showing terminal spores. (After Kolle and Wassermann.) 


gram of brain substance suspended in salt solution is able to neu- 
tralize 10 fatal doses of tetanus toxin, forming a loose combina- 
tion from which the toxin may be set free by drying. Most 
mammals are very susceptible, although cats and dogs are only 
slightly so. Birds are relatively resistant and some reptiles are 
wholly refractory to the tetanus toxin. ; 

Von Behring and Kitasato in 1890 produced immunity in 
rabbits, and later in horses, by injecting into them toxin to which 
iodine trichloride had been added, and subsequently unaltered 
toxin. The immunized animal was able to survive an injection 


? Von Lingelsheim, Kolle and Wassermann, Handbuch, 1912, Bd. IV, S. 766. 


BACILLACEH! THE SPOROGENIC ANAEROBES 203 


many times: greater than the amount necessary to kill a normal 
animal. Moreover, the cell-free blood serum of the immunized 
animal was found to neutralize the poison in a test-tube and to 
protect a normal animal against fatal 
doses of it. The new substance of the 
blood capable of rendering the toxin 
harmless was ‘called antitoxin. One 
antitoxic unit of tetanus antitoxin, ac- 
cording to Von Behring is the amount 
which will neutralize 40 million times | 
-the amount of fresh tetanus toxin 
_ necessary to kill a mouse weighing 15 
grams (40 million X the 15 + Ms 
dose) so completely that only a slight 
local contraction, indicated by a fold- 
ing of the skin, results from sub-. 
cutaneous injection of the mixture into 
a mouse (the L, effect). This amount 
of toxin (40 million X the 15 + Ms 
dose) is generally measured in practice 
against a standard antitoxin and is 
designated as a toxic unit. The toxin 
is preserved in a dry state. To test a 


of a 


new antitoxin one employs 


IO0o0 
oxic uni fe} the 1 Ms Fic. 117.— Clostridium tetani. 
t HEI (40,00 x 5 ss Stab culture in glucose gelatin, 


dose) and ascertains the amount of six days old. (From McFarland 
serum which must be added so as to , %/@” Fraenkel and Pfeifer.) 

neutralize it to the L. end point. Each trial mixture is 
diluted to 1 c.c. with salt solution and 0.25 c.c. per 10 grams of 
body weight is injected into a mouse. When the typical Lo effect 
is produced in the mouse, the amount of antitoxic serum employed 
in the preparation of this particular mixture is said to represent 


ace antitoxic unit. Ordinarily the mixture of toxin and anti- ° 


2904 SPECIFIC MICRO-ORGANISMS 


toxin is allowed to stand 30 minutes before injection. Comparable 
results are obtained only by following a definite procedure and it 


% 7 
is especially necessary to use the conventional dose of 
; 1000 


antitoxic unit and Ee toxic unit in the standardization of sera. 


The standard unit employed in the United States is some- 
what different from the Von Behring antitoxic unit. The Ameri- 
can immunity unit of tetanus antitoxin is ten times the least 
amount of antitetanic serum necessary to preserve the life of a 


Fic. 118.—Clostridium botulinum. Some individuals containing spores. (After van 
Ermengem.) 


guinea-pig weighing 350 grams for 96 hours against the official 
test dose of standard tetanus toxin furnished by the Hygienic 
Laboratory of the U. S. Public Health Service. Tetanus anti- 
toxin deteriorates with moderate rapidity. The reaction be- 
tween tetanus toxin and antitoxin seems to take place in two 
stages, first a reversible absorption and following this a specific 
chemical union. 


1 Rosenau and Anderson: U. S. Hygienic Laboratory, Bulletin No. 43, 1908, p. 59- 
The official test dose of toxin is roo times the amount of a dry tetanus toxin required 
to kill a 350 gram guinea-pig in four days. 


BACILLACEM: THE SPOROGENIC ANAWROBES 295 


Tetanus antitoxin seems to be a reliable preventive of teta- 
nus if given soon after the wound is inflicted in a dose of 20 anti- 
toxic units (German) or 1500 immunity units (U. S. Standard). 
After symptoms of tetanus have appeared, antitoxin is of less 
use. At this time the poison is present not only in the vicinity 
of the wound and in the blood but also in the peripheral nerves 
and in the central nervous system. The toxin in the last two situ- 
ations is only slightly or not at all influenced by subcutaneous in- 
jection of antitoxin. That in the peripheral nerves may be 
reached by intraneural injection, and in subacute or chronic cases 
recovery may sometimes take place. Acute cases in which symp- 
toms appear in a few days after infliction of the wound offer no 
hope. Prophylactic use of tetanus antitoxin in all punctured and 
lacerated wounds, especially those caused by gunpowder (Fourth 
of July) is an essential feature of the effective treatment for tet- 
anus. Surgical cleansing and antiseptic open treatment of such 
wounds is to be recommended.? 

Late tetanus may appear even after the antitoxin has been 
given early, but in such cases the disease is usually milder in char- 
acter and may be successfully treated by further antitoxin and 
surgical attention to the wound. 

Clostridium Botulinum.—Van Ermengem in 1895 discovered 
the spores of this organism in the intermuscular connective tissue 
of a ham which had given rise to 30 cases of food poisoning with 
3 deaths. Othér anaérobic as well as aérobic bacteria were also 
present in the meat. Its natural habitat is unknown but it seems 
to occur in the feces of swine. The bacillus 0.9 to 1.24 wide 
by 4 to 6u long and occurs single or in pairs. It is slightly motile 
and has 4 to 8 peritrichous flagella. It is Gram-positive. The 
spores are oval and usually nearer one end of the cell. They are 
quite variable in resistance. Van Ermengem found that they were 
killed at 80° C. in 30 minutes and by boiling for 5 minutes. More 
recent careful tests by Burke? have shown that the spores may 


1 Editorial, Jour. A. M. A., 1909, Vol. LIII, p. 955. 
2 Burke, Journ. Amer. Med. Assn., 1919, 72, p. 88. 


‘ 
@ 


. 296 SPECIFIC MICRO-ORGANISMS 


sometimes resist. boiling in water for 2 hours and autoclave heat 
-at 5 pounds pressure for ro minutes. 

‘ Strict anaérobiosis is necessary for successful sultare, except 
when B. botulinum grows in symbiosis with aérobes. Growth is 
best at 25-30” C., very slight at 37°-38.5° C., and best in a medium 
slightly alkaline to litmus. Gelatin is quickly liquefied and 
abundant gas is produced in glucose media. The organism ap- 
pears to be incapable of growth in the animal body. Cultures 
are very poisonous when injected into or fed to animals. 

The poison ‘“Botulin” resembles in some of its properties the 
tetanus toxin. It is destroyed rapidly at 70°-80° C., and pre- 
serves its toxicity for years when dried. It is neutralized by 
mixing with brain substance. It differs from the other 'pow- 
erful toxins, however, in its ability to resist the gastric juice and 
to poison by absorption through the alimentary canal. Forssman 
has immunized guinea-pigs, rabbits and goats, and has obtained 
an antitoxic serum from these animals. rs 

Botulism is a form of food poisoning definitely recognized as 
such as early as 1820. The symptoms are very characteristic, 
appearing in 18 to 48 hours after ingestion of the poisonous food. 
There is vomiting, dryness of the mouth and constipation, motor 
paralysis, especially early in the external ocular muscles. The 
involvement of the central nervous system may progress to com- 
plete motor paralysis and death. The mind is usually clear even 
in the fatal cases. The early outbreaks of the disease followed 
the consumption of sausage, hams, fish and other cured or pre- 
served meats. More recently outbreaks of botulism have been 
recognized in the United States with increasing frequency and 
their causation has been traced not only to meat foods but to . 
various canned vegetables and even fruits. Dickson! and his 
associates have shown that commercial canners processes as well 
as home canning methods cannot be relied upon to kill the spores 
of Cl. botulinum if this organism happens to be present in the raw 


? Dickson, Burke and Ward: Archives of Internal Med., 1919, 24, p. 581. Refer- 
ences to literature are given in this paper. 


BACILLACE&: THE SPOROGENIC ANAEROBES 297 


material. All canned food, which shows the slightest evidence of 
spoilage, should either be discarded or else boiled before it is 
eaten, as botulin is destroyed by boiling. ; 

Forage poisoning in domestic dnimals has been shown to be 
due to Cl. botulinum by Graham! and his associates. 


1 Graham and Brueckner: Studies in forage poisoning, Jowrn. of Bact., 1919, 4 
p. 1. References to literature are given in this paper. 


CHAPTER XIX 


MYCOBACTERIACE#: THE BACILLUS OF DIPHTHERIA 
AND OTHER SPECIFIC BACILLI PARASITIC ON 
SUPERFICIAL MUCOUS MEMBRANES 


Bacillus (Corynebacterium) Diphtheriz.—Klebs in 1883 dis- 
covered this organism in the microscopic study of pseudomem- 
branes from fatal cases of epidemic diphtheria. Léoffler in 1884 
obtained pure cultures of the bacillus and by inoculating the 
abraded mucous membrane of susceptible animals with his cul- 
tures, he produced local lesions similar to those observed in human 
diphtheria, in some instances followed by death or paralysis. 

B. diphtherie occurs in the exudate (false membrane) which 
occurs in the pharynx, larynx and adjacent mucous membranes 
in epidemic diphtheria, on the mucous membranes of those who 
have recovered from the disease and, much less commonly, on 
the mucous membranes of healthy throats. It is a rod-shaped 
organism extremely variable in size, shape and staining properties. 
The width is ordinarily between 0.3 and 0.84 and the length 
varies from 1 to 64. The cell is straight or slightly curved and 
very frequently of uneven diameter, with swelling at one end or 
in the middle portion. The cell contents stain unevenly in 
many of the cells. Many different morphological types are thus 
presented which may be designated roughly as regular cylinders, 
clubs, spindles and wedges according to form, and as uniformlv 
pale, uniformly dark, regularly or irregularly banded or granular 
according to internal structure of the stained cell. These varia- 
tions in form and internal structure are best seen after staining 
the bacillus. with Loffler’s methylene blue and are especially 
valuable in the quick recognition of B. diphtherie as it grows in 
the diphtheritic membrane or in culture on Léffler’s blood serum. 

208 


4 


MYCOBACTERIACEZ: THE-BACILLUS OF DIPHTHERIA 299 


On other media, such as glycerin agar, the morphological irregulari- 
ties are less marked as a rule. The oganism in young cultures 


Fic. r19.—Bacillus of diphtheria. 1000. 


' Fig. 120.—B. diphtheria stained by Neisser’s method. 


stains readily, best perhaps with Léffler’s methylene blue in 
the cold. It is Gram-positive.» Old cultures stain with great 
. difficulty. 


300 


Léffler’s blood serum is the medium of choice. 


SPECIFIC MICRO-ORGANISMS 


The colonies 


develop at 37° C. in 8 to 12 hours as grayish, slightly elevated 
points and become 2 to 3 mm. in diameter in the course of 48 


fon 


_ Fic. 121.—Forms of B. diphtherie in cultures on Léffler’s serum. A, Charac- 
teristic clubbed and irregular shapes with irregular stcining of the -cell contents. 


x<1100. B, Irregular 
Williams.) 
-hours. 


shapes with even staining. 


Contiguous colonies become confluent. 


x1000. (After Park and 


On glycerin agar 


after 25 hours at 37” C., the colony is coarsely granular with 
somewhat jagged outline. Many variations from this typical 


Fic. 122.—Forms of B. diphtherie in cultures on agar. A, Bacilli small and 
B, Spherical forms in culture 24 hours old. On Léffler’s serum 


uniform. X1000. 


this same organism produced granular forms. 


appearance occur. 


XI4I10. 


Growth in gelatin is slow and ceases below 
20° C. The medium is not liquefied. The bacillus grows in 
milk without producing coagulation. In broth the growth 


(After Park and Williams.) 


MYCOBACTERIACE#: THE BACILLUS OF DIPHTHERIA 301 


may occur as a granular sediment, as a diffuse cloudiness or as a 
pellicle on the surface, depending upon the reaction and pepton 
content of the medium and the vigor of growth of the culture. 
The growth on the surface produces the best yield of toxin. Acid 
is produced in dextrose broth. The organism is killed when moist 
by heating to 60° C. for 20 minutes. It is fairly resistant to 
drying and has been found alive in bits of dry diphtheritic mem- 
brane after four months. 

_ Roux and Yersin in 1888 filtered vee cultures. of the diph- 
theria bacillus through porcelain filters and found the filtrate 


Fic. 123.—Colonies of B. diphtheri@ on agar. X200. (After Park and Williams.) 


extremely poisonous. By injecting it into animals they were 
able to produce the signs of local and general intoxication which 
are observed in the natural disease. A favorable medium for 
toxin production is a veal broth containing 2 per cent pepton. 
and having a titre of 9 c.c.1 of normal sodium hydroxide above 
the neutral point to litmus. It should be placed in flasks in a 
thin layer to allow abundant air supply. Incubation for from 
5 to 10 days gives the maximum toxicity. The filtrate from such 

a culture may kill a 250 gram guinea-pig in a dose of 0.002 c.c 

Less powerful toxin is frequently obtained, so that sometimes 


‘Per 1000 c.c, of the medium. 


302 SPECIFIC MICRO-ORGANISMS 


even 0.5 c.c. or more may be required to kill a guinea-pig, and 
some strains of bacilli morphologically indistinguishable from 
B. diphtherie seem to produce no toxin at all. The toxin is 
quickly destroyed by boiling and 
loses g5 per cent of its strength in 
five minutes at 75° C. It gradually 
deteriorates even at low tempera> 
tures. Its chemical nature is un- 

known. Ehrlich has shown that old 

toxin which has lost much of its 

poisonous property is still able to 

combine with as much antitoxin as 

before. This deteriorated toxin is 

called toxoid. He explains the phe- 

nomenon by assuming the existence 

of two distinct chemical groups in the 
toxin molecule, one serving to com- 

bine with antitoxin and being rela- 

tively stable, the other bearing the 

poisonous properties and readily un- 

dergoing disintegration. The former 

he has called the haptophorous group 

and the latter the toxophorous 

group. In toxoid the toxophorous 

group has degenerated. 

Diphtheria was recognized as a i 
distinct disease by Bretonneau in 
1821. It is characterized by a local 
inflammation, usually on the mucous 


Fic. 124.—B. diphtheria, culture : 
‘oni plycerine daar, membrane of the throat, the nose, 


more rarely the genital mucous 
membrane, or the surface of a wound, and by an accompany- 
ing general intoxication giving rise to focal necrosis in various 
parenchymatous organs and affecting more particularly the 
heart and the nervous system. The local inflammation may 


MYCOBACTERIACEH: THE BACILLUS OF DIPHTHERIA 303 


be only a mild reddening or it may be a widespread ‘area of 
necrosis. Most frequently there is an exudate of plasma con- 
taining leukocytes, epithelial cells and bacteria, and this coagulates 
on the mucous surface. The epithelium underneath also under- 
goes necrosis in moderately severe cases and is firmly attached to 
the exudate by the fibrin threads. In severer 
forms there is an escape of blood into the exudate 
giving it a dark color. The local lesion is largely 
due to soluble toxin formed by the bacilli. The 
general disturbance is, as a rule, due solely to the 
absorbed toxin. The bacilli remain at the site of 
the lesion and do not appear in the blood or in- 
ternal organs in any appreciable numbers. They 
are occasionally found in the spleen or kidney of 
fatal cases, but not more frequently than the 
streptococcus is found in these organs in ap- 
parently uncomplicated fatal cases of diphtheria. 

The local lesion in the throat may be simu- 
lated very closely by inflammation due to 
streptococci, but the general manifestations are 
not duplicated in such conditions. Mixed in- Zo ee 
fection with diphtheria bacilli and virulent used in the’ diag- 

2 oe « nosis of diphthe- 
streptococci may present a clinical picture of yi. The pledget 
great severity. Bacteriological examination is °f cotton on the 

3 a A . wire shown is 
often a great help in diagnosis even to the expert much too bulky. 
clinician, and is quite generally employed. 

Bacteriological Diagnosis of Diphtheria.—In many large cities 
the bacteriological diagnosis of diphtheria is undertaken by 
boards of health. The methods used differ somewhat in detail, 
but are similar in the main, and are based upon the procedure 
devised by Biggs and Park for the Board of Health of New York 
City. Two tubes are furnished in a box. The tubes are like 
ordinary test-tubes, about three inches in length, rather heavy and 
without a flange. Both are plugged with cotton. One contains 
slanted and sterilized Loffler’s blood-serum mixture (Fig. 125); 


304 SPECIFIC MICRO-ORGANISMS 


the other contains a steel rod, around the lower end of which a 
pledget of absorbent cotton has been wound. These tubes con- 
taining the swabs are sterilized. The swab is wiped over the 
suspected region in the throat, taking care that it touches nothing 
else, and is then rubbed over the surface of the blood-serum mix- 
ture. The swab is returned to its test-tube and the cotton plugs 
are returned to their respective tubes. The plugs, of course, 
are held in the fingers during the operation, and care must be 
taken that the portion of the plug that goes into the tube touches 
neither the finger nor any other object. The principles, in fact, 
are the same as those laid down in general for the inoculation 
of culture-tubes with bacteria (see page 111). In board-of-health 
work these tubes are returned to the office. When it is desirable, 
a second tube may be inoculated from the swab. The tubes 
are placed in the incubator, where they remain for from 6 to 15 
hours and a microscopic examination is then made of smear 
preparations stained with Loffler’s methylene blue. After use 
the tubes and swabs should be most carefully and thoroughly 
sterilized. 

On Léffler’s blood-serum kept in the incubator the bacillus 
of diphtheria grows more rapidly than most other organisms 
which are ordinarily encountered in the throat, a property which 
to a certain extent sifts it out, as it were, from them, and makes 
its recognition with the microscope easy in most cases. The 
appearance of the bacilli under the microscope is quite charac- 
teristic. The diagnosis of the diphtheria bacillus in practice is 
made from the character of the growth upon the blood-serum and 
the microscopical examination, taking into account the size and 
shape of the bacilli, with the frequent occurrence of irregular. - 
forms and the peculiar irregularities in staining, and this usually 
-suffices; but in doubtful cases a second culture should be made 
from the throat, and at the same time another tube of Loffler’s - 
serum should be inoculated from the first culture. On the next 
day plate cultures on glycerin agar may be made, from which 
typical colonies should be transplanted to broth. . After 48 hours 


MYCOBACTERIACEH: THE BACILLUS OF DIPHTHERIA 305 


at 37° C. the broth is injected into two guinea-pigs in doses of 
0.5 c.c. and one of the guinea-pigs should receive at the same time 
diphtheria antitoxin. In this way virulent diphtheria bacilli 
may be accurately detected. . : 

-The very large number of examinations that have been made 
by various boards of health have shown that the diphtheria 
bacillus may persist in the throat for a long time—occasionally 
several weeks after the patient has apparently recovered; also 
that diphtheria bacilli are occasionally found in the throat, when 
there is an inflammatory condition without any pseudo-membrane 
and that they not only appear in an apparently healthy throat, 
especially in hospital nurses and in children who have been asso- 
ciated with cases of diphtheria, but also in those who have had 
no traceable contact with diphtheria cases.1_ It has been found 
that bacilli sometimes occur in the throat, which have all the 
morphological and cultural properties of the diphtheria bacillus, 
but which are devoid of virulence when tested upon animals. 
Such diphtheria bacilli have frequently been called pseudodiph- 
theria bacilli. A bacillus closely resembling the diphtheria bacillus, 
but without virulence, has been found in xerosis of the conjunctiva. 
It is called the xerosis bacillus. If not a transformed diphtheria 
bacillus, it is at least closely related. The diphtheria bacillus is 
subject to wide variations in morphology, so that, in dealing with 
unknown cultures where the forms are not characteristic and in- 
jection into animals is without result, it may be difficult to decide 
whether or not the organisms are diphtheria bacilli. 

The disease is undoubtedly transmitted very largely by 
immediate contact, especially with persons harboring the bacilli 
but not seriously ill, and by fomites. Children in school or at 
play readily transfer secretions of the mouth, ‘and a cough or 
sneeze may distribute such material over a wide area. 

- Immunity to diphtheria was produced by Von Behring in 
1890 by injecting the toxin into animals, the general method of 
- procedure being quite similar to that followed in the production 


1Sholly: Journ. Infect. Dis., Vol. TV, 1907, pp. 337-346. * 
20 


306 SPECIFIC MICRO-ORGANISMS 


of tetanus antitoxin. The blood serum of the immunized animal 
was found to be capable of neutralizing the poisonous property 
of diphtheria toxin. The brilliant success of Roux (1884) in treat- 
ing diphtheria with antitoxic serum caused the rapid adoption 
of antitoxin as a therapeutic agent throughout the world. Park 
and his co-workers, Atkinson, Gibson and Banzhaf, have devel- 
oped a method of concentrating diphtheria antitoxin which is 
now generally employed. 

For the production of antitoxin' young healthy horses are 
selected with great care. They are specifically tested for tubercu- 
losis and glanders. A powerful diphtheria toxin is then injected 
into the horses, in an amount of sufficient to kill 5000 guinea-pigs, 
together with 10,000 units of antitoxic serum. The toxin is 
subsequently injected at intervals of three days and each succeed- 
ing dose is increased by about 20 per cent as long as the condition 
of the horse is satisfactory. At the end of two months the dose 
is about fifty times as large as the initial dose. Antitoxin is 
given only at the start. The serum of the horse is tested from 
time to time and, when the desired antitoxic strength has devel- 
oped, the blood is drawn once a week for the preparation of anti- , 
toxin. A dose of toxin is given after each weekly bleeding. 
The blood is drawn from the jugular vein into jars containing a 
10 per cent solution of sodium citrate, nine parts of blood to one 
of the citrate solution. The material is mixed and allowed to 
sediment in a refrigerator. The plasma is then siphoned off 
into large bottles and heated to 57° C. for 18 hours to change 
part of the soluble globulins” to euglobulins, insoluble in a satu- 
rated solution of sodium chloride. An equal volume of saturated 
aqueous solution of ammonium sulphate is then added. The 
precipitate which forms consists of the globulins and nucleopro- 
teins of the plasma. ‘This precipitate is collected on a filter 


1 For details of the method see Park and Williams, Pathogenic Bacteria and 
Protozoa, Phila. 

2 Banzhaf: The Preparation of Antitoxin; Johns Hopkins Hosp. Bull., 1911, 
Vol. XXII, pp. 106-109, 


MYCOBACTERIACEA: THE BACILLUS OF DIPHTHERIA 3047 


and extracted with a saturated solution of sodium chloride, in 
which the pseudoglobulin fraction, carrying with it the antitoxic 
property, is dissolved: This is precipitated by the addition of 
dilute acetic acid, filtered out and again taken up in salt solu- 
tion. It is carefully neutralized with sodium carbonate and 
dialyzed for several hours against water to remove the inorganic 
salts. - The residue in the dialyzer is then passed through a 
Berkefeld filter to sterilize it, a preservative is added, and it is 
ready to be tested and put up in. containers for distribution. 
The final product contains 75 to go per cent of the original anti- 
toxic strength and is only about one-third as bulky. The serum 
albumin, euglobulin and nucleoprotein have also been to a large 
extent eliminated in the process of concentration. 

The antitoxic strength of anti-diptheritic serum is expressed 
in immunity units and is ascertained by animal experimentation. 
The von Behring unit is contained in ten times the amount of 
serum required to protect a 250 gram guinea-pig perfectly from 
"the effects of ten times the dose of fresh diphtheria toxin which 
kills a similar guinea-pig in four days. The dose of toxin is 
first ascertained by trial on guinea-pigs and the dose necessary 
to kill in four days (minimum lethal dose) determined. Ten 
times this quantity is then injected along with varying doses of 
antitoxic serum into a series of guinea-pigs until the quantity 
of serum, which not only saves the animal but prevents loss of 
weight and local induration at the site of injection, has been 
ascertained. Ten times this amount contains one immunity unit. 

Ehrlich has carefully standardized an antitoxic serum and 
has preserved it as a dry powder, of which one gram contains 
1700 immunity units. This standard is now employed as the 
official standard for comparison in the United States. In stand- 
ardizing an antitoxin by the Ehrlich method, one unit of the 
standard antitoxin is injected along with various quantities of a 
toxin to ascertain how much of the latter is required so that the 
animal dies after four days. This dose of toxin, which when 
combined with one unit of the standard antitoxin, kills a 250 


308 SPECIFIC MICRO-ORGANISMS 


gram guinea-pig in four days is called the L; dose. One next 
injects this L, dose along with varying quantities of the new 
antitoxin, and the amount of the latter which keeps the guinea- 
pig alive for just four days, or, in other words, produces the same 
effect as the standard unit, is known to contain one immunity 
unit. In the United States, the Hygienic Laboratory at Washing- 
ton furnishes standard antitoxin to manufacturers for this official 
test and all marketed sera are tested by this method. 

Diphtheria antitoxin not only prevents the development of 
diphtheria when injected in doses of 1000 units, but it also exerts 
a marked influence as a therapeutic agent in diphtheria, neutraliz- 
ing the poison produced by the bacilli in the body of the patient. 
It does not kill the bacilli but it nullifies their chief offensive 
weapon, the soluble diptheria toxin. Its value in treatment 
of diphtheria is everywhere attested by clinical evidence. The 
inflammation in the throat subsides and the membrane disappears. __ 
The bacilli, however, may remain for a considerable time. Local 
antiseptics may assist the natural agencies of the body in their — 
destruction. In some cases they persist for months in spite of 
vigorous treatment. 

Certain untoward effects have followed the injection of anti- 
diphtheritic serum. Sudden death -has occurred in very rare 
instances and skin rashes are rather common. These effects 
are probably due to toxic substances set free in the. parenteral 
digestion of the foreign protein and’ are doubtless of the same 
general nature as the phenomenon of anaphylaxis. Since the 
introduction of the concentrated antitoxin fatalities-have become 
exceedingly rare or have been entirely eliminated. The serum 
rashes and cases of nervous shock do occur, especially in asthmatic 
individuals and in those who have received a previous injection 
of horse serum. In these persons it is well to give a minute 
quantity, o.2 c.c., of the serum as a preliminary injection, wait 
two or three hours and then give the full dose. The danger of 
serious reactions due to anaphylaxis may thus be avoided. 

1 Vaughan: Amer. Journ. Med. Sciences, 1913, Vol. CXLV, pp. 161-177. 


MYCOBACTERIACE&: THE BACILLUS OF DIPHTHERIA | 309 


The Schick Reaction—In an outbreak of diphtheria it is often 
helpful to ascertain which of the exposed persons may be sus- 
ceptible to the disease. For this purpose the diphtheria toxin 
is diluted with salt solution so that 0.2 c-c. of the solution contains 
1é9 M. L. D. (minimum lethal dose fora 250-gram guinea Pig). 
This amount is injected through a fine needle info the person’s 
skin so that a white swelling is produced. A control injection may 
be made in another place with the same toxin previously rendered 
inert by heating at 75° C. for 5 minutes. A positive reaction is 
manifested by a red, swollen and infiltrated area 7 to 20 mm.in 
diameter after 48 to 96 hours at the test area without a similar 
reaction at the control site and this result indicates that the 
individual is susceptible to diphtheria. : 

Bacillus (Corynebacterium) Xerosis.—This organism occurs 
on the normal mucous membranes, particularly the conjunctiva. 
It resembles B. diphtherie very closely, simulating the granular 
morphological type. Its cultures are not poisonous. 

Bacillus Hofmanni.—This organism is also called the pseudo- 
diphtheria bacillus. It occurs frequently in cultures from the 
nose and pharynx, and resembles the short solid-staining morpho- 
logical types of B. diphtheri@. It does not produce toxin, nor 
does it produce acid from dextrose. ss 

The Morax-Axenfeld Bacillus.—This is a small non-motile 
diplo-bacillus, the individuals measuring about 1 X 2u, which 
occurs in one form of epidemic conjunctivitis. It can be cultured 
on Léffler’s serum, which it liquefies, and the disease has been 
produced in man by inoculation with pure cultures. 

The Koch-Weeks Bacillus.—This a non-motile rod 0.25 
wide and 1 to 2m long, which occurs in epidemic conjunctivitis. 
It is cultivated with difficulty . and abundant moisture is essential 
to success. Inoculation with pure cultures causes conjunctivitis. 

Bacillus (Hemophilus) Pertussis (Bordet-Gengou Bacillus).— 
Bordet and Gengou in 1906 described a minute, non-motile 
bacillus 0.3. X 1.54 which occurs in the sputum and on the mucous 
membrane of the trachea and bronchi in whooping cough. They 


“ 


310 SPECIFIC MICRO-ORGANISMS 


obtained cultures of the organism on blood agar and, employing 
these cultures as an antigen, they demonstrated an antibody 
in the blood of patients by means of the complement-fixation 


Fic. 126.—The Morax-Axenfeld bacillus in the exudate of conjunctivitis. (From 
McFarland after Rymowiisch and Matschinsky.) 


est. Klimenko! has further succeeded in producing a chronic 
catarrh of the respiratory passages in monkeys and puppies by 


Fic. 127.—Koch-Weeks bacillus in muco-pus from conjunctivitis. 1000. (From 
Park and Williams after Weeks.) 


applying pure cultures to the tracheal mucosa. The bacillus 
is a minute rod, motionless, stained with moderate difficulty, 
LCentralbl. f. Bakt. Orig., 1909, Bd. XLVIII, S. 64-76. 


MYCOBACTERIACEA: THE BACILLUS OF DIPHTHERIA 311 


and Gram-negative. It occurs in large numbers between the 
cilia of the epithelial cells lining the trachea and bronchi in cases 
of whooping cough where it mechanically! interferes with the 
action of the cilia and gives rise to irritation. It is an obligate 
aérobe and at first grows well only on media containing blood, 
ascitic fluid or other protein. Later it adapts itself to artificial 
culture on ordinary media. Gelatin is not liquefied. : 

Bacillus (Hemophilus) Influenzz.—Pfeiffer in 1892 isolated a 
small bacillus 0.254 wide by 0.5 to 2.04 long from the bronchial 
secretion in cases of epidemic influenza. The bacillus occurs in 
enormous numbers in acute uncomplicated cases of influenza 
in the nasal and bronchial mucus. It is non-motile, aérobic, 
rather difficult to stain and Gram-negative. Cultures are ob- 
tained on ordinary agar smeared with fresh human or rabbit’s 
blood or upon a mixture of blood and agar. Hemoglobin seems . 
essential to growth. The bacillus is very sensitive to drying, 
and its transmission would seem to occur largely through close 
association, and the scattering of moist droplets of material 
from the nose and mouth in sneezing, coughing and talking. 
The cultures are toxic for rabbits and monkeys. The causal 
relation of B. influenze to influenza is not as yet fully established. 
Conditions resembling influenza very closely seem to be caused 
by other organisms, such as the cocci. 

The influenza pandemic of 1918 has stimulated numerous 
investigations of the disease but the bacteriology of it has not 
been fully elucidated.? 


e 

1 Mallory: Pertussis: The Histological Lesion in the Respiratory Tract, Journ. 
Med. Rsch., 1912, Vol. XXVII, pp. 115-124; Mallory, Hornor and Henderson, 
Journ. Med. Rsch., 1913, Vol. XXVII, pp. 391-397- 

2 Park: Bacteriology of recent pandemic of influenza and complicating infections, 
Journ. Amer. Med. Assn., 1919, 73,P- 318; Huntoon and Hannum: Rdle of Bacillus 
influenze in clinical influenza, Journ. of Immunology, 1919, 4, Pp. 167; MacNeal: 
The influenza epidemic of 1918 in the American Expeditionary Forces in France 
and England, Archives Int. Med., 1919, 23, p. 657; Blake and Cecil: The production 
of an acute respiratory disease in monkeys by inoculation with Bacillus influenze, 
Journ, A. M. A., Jan. 17, 1920, 74, P. 170. 


312 : SPECIFIC MICRO-ORGANISMS 


Bacillus (Bacterium) Chancri (Bacillus of Ducrey).—Ducrey 
in 1889 found a short bacillus in the soft venereal sore known 
as chancroid, obtained it in pure culture and produced typical 
lesions by inoculation in man. The organism is about 0.5 X 1.5y, 
often growing in threads. It grows on a blood-agar mixture at 
37° C. Material for culture should be obtained from an un- 
broken pustule or from a chancroidal bubo, so as to avoid 
contaminating organisms. The bacillus possesses very little resist- 
ance to drying or to germicides. Successful inoculation experi- 
ments have been carried out on man, on monkeys and on cats. 
Other organisms! appear to produce soft chancre in the absence 
of the Ducrey bacillus in some cases. 


' Herbst and Gatewood: Journ. A. M. A., 1912, Vol. LVIII, pp. 189-191. 


CHAPTER XX 


MYCOBACTERIACE: THE TUBERCLE BACILLUS AND 
OTHER ACID-PROOF BACTERIA 


Bacillus (Mycobacterium) Tuberculosis.— Robert Koch in 1882 
discovered the minute rods in tuberculous tissue, planted the 
tissue on slanted inspissated blood serum and obtained :pure 
cultures of the tubercle bacillus, inoculated these cultures: into 
animals and produced typical tuberculosis. He succeeded in 
doing this with natural tuberculosis of man and many other 
mammals and also with the tuberculosis of birds. Silbey in 
1889 observed with the microscope morphologically similar 
bacilli in a snake. Rivolta and Mafucci in 1889 pointed out the 
differences between the tubercle bacillus of birds and that of 
mammals and their work, together with subsequent confirmatory 
investigations, has established a distinct avian type of tubercle 
bacillus, B.. tuberculosis var. gallinaceus. In 1897 Bataillon, 
Dubard and Terre found acid-proof bacilli in definite histological 
tubercles in a fish (carp), obtained cultures and recognized ‘it as 
distinct from the mammalian form, and it was subsequently 
designated as B. tuberculosis var. piscium. Theobald Smith 
in 1898 published the results of a careful and extensive com- 
parative study of tubercle bacilli from human sputum and 
tubercle bacilli from tuberculous tissue of the bovine pearl 
disease (tuberculosis), and pointed out distinct differences in 
morphology, cultural characters and virulence between the 
organisms derived from the two sources. The mammalian 
tubercle bacilli were thus divided into two types, and subsequent 
investigation has. fully justified the recognition of B. tuberculosis 

313 é 


314 SPECIFIC MICRO-ORGANISMS 


var. humanus and B. tuberculosis var. bovinus. Some, or perhaps 
all four of these types may be eventually recognized as distinct 
species. At present the designation as types or varieties seems 
more appropriate. : 

Bacillus Tuberculosis var. Humanus.—This organism occurs 
in the infiltrated lung in human phthisis and also in the great 


Fic. 128.—Bacillus tuberculosis in the sputum of a consumptive; stained by Ziehl 
method. X2100. (After Kossel.) 


majority of the other tuberculous lesions in man. In the ex- 
ternal world it does not grow naturally and passes there a more 
~ or less temporary existence in discharges from the body, of which 
the most important is the sputum. The cell is about 0.4m in 
width and quite variable in length, 0.5 to 8.ou. ‘The longer 


MYCOBACTERIACE#: THE TUBERCLE BACILLUS 315 


forms are often somewhat bent, and they frequently contain 
refractile granules. When stained these forms have a beaded 
or banded appearance. Spores have not been observed. Branch- 
: ing forms occur sometimes in cultures, suggesting a close relation 
to actinomyces and streptothrix. There is a considerable amount — 
of a waxy substance in the body of the bacillus, which makes it 
difficult to stain and also difficult to decolorize after it has been 
stained. Hot carbol-fuchsin is generally employed, applying 
it for one to two minutes. The preparation is then washed and 
decolorized in dilute mineral acid (2 to 20 per cent) and in alcohol. 


Fic. 129.—Bacillus tuberculosis, from a pureculture. X 1000. - 


Tissue elements and most other materials may be completely 
bleached by this treatment, leaving the tubercle bacilli still 
colored. B. tuberculosis is Gram-positive. 

Cultures are most readily obtained by transferring bits of 
tuberculous tissue, free from other micro-organisms, to moist 
slants of inspissated blood serum or Dorset’s egg medium. If 
the available material is already contaminated, the extraneous 
organisms may usually be eliminated by inoculating uit into 
guinea-pigs and making the cultures from the tuberculous guinea- 


31 6 SPECIFIC MICRO-ORGANISMS 


pig tissue, about four weeks later. The tubes may be sealed 
with rubber caps or paraffin and incubated at 37° C. Better 
results are obtained by leaving the tubes unsealed and incubating 
at 37° C. in an atmosphere saturated with moisture, as the bacillus ° 
is a strict aérobe, but this requires special care and is not absolutely 
essential to success. After two or three weeks a dry, white growth 
is developed which may later become folded. Transplants 
from the ‘primary culture to. glycerin agar, glycerin broth or 


Fic. 130.—Tubercle bacillus showing branching and involution forms. (After 
Migula.) 


glycerin potato are usually successful. Old cultures on potato 
and agar often become yellowish oreven pink in color. 

The chemical composition of tubercle bacilli has been ex- 
tensively studied. The moisture content varies from 83 to 89 
per cent. The ash (inorganic salts) amounts to about 2.6 per 
cent of the dry substance, and about half of this is phosphoric 
acid P04, The waxy constituent of the bacterial cells is of 


1Tt is possible to cultivate tubercle bacilli directly from contaminated material 
such as sputum, by careful technic, although the results are somewhat uncertain: 
A method is described in detail by Williams, W. W. and Burdick, Journal of Bact.,. 
1916, I, p. 411-414. 


MYCOBACTERIACEZ: THE TUBERCLE BACILLUS 317 


S 
particular interest. This makes up from 
8 to 40 per cent of the dry substance, less 
in young and more in old cultures. The 
acid-proof staining property depends upon 
this waxy substance, for the bacilli from 
which it has been extracted by ether- 
alcohol are no longer acid-proof while the 
wax itself exhibits this peculiarity of 
staining. It is also known that the bacilli 
in young cultures are on the whole less 
acid-proof than those from old cultures in 
_which chemical analysis shows a greater 
concentration of the waxy substance. The 
protein substances, largely nuclein, make 


up about 25 per cent of the dry cell sub-_ 


stance. Several other constitutents of the 
cell have been identified. As in the case 
of other bacteria the chemical composition 
varies within rather wide limits according 
to the nutritive medium, conditions of 
growth and especially the age of the 
culture. 

The poisons of the tubercle bacillus 
exist to a large extent in an inactive form 
in the culture fluid and more particularly 


as an undissolved constituent of the 


bacterial cell bodies. Culture filtrates 
exert little or no effect upon injection into 
normal animals. The dead bacilli, how- 
ever, give rise to local inflammation and 
in many instances stimulate the formation 
of typical tubercles at the point where 
they lodge. Evidently the poison is set 
free from some substance in the dead 
cells by the action of the tissue cells or 


Fic. 131.—Bacillus tuber- 
culosis. Culture on glycerin 
agar several months old. 
(From McFarland after 
Curtis.) 


31 8 SPECIFIC MICRO-ORGANISMS 


body fluids upon them, and it is quite certain that the bacteria- 
free culture fluid (old tuberculin) becomes toxic as a result of such 
an action. 

Tubercle bacilli outside the body are moderately resistant 
to harmful influences. In dried sputum, they have been found 
alive after eight months. Direct sunlight kills the bacilli in 
sputum in a few minutes if this be exposed in a thin transparent 
layer. In thicker masses the effect of light is uncertain. “In 
buried cadavers the bacilli remain alive and virulent for 2 to 6 
months. In watery suspensions the bacilli are killed by heating 
to.60° C. for 15 minutes. In milk, heating at 60° C. for 20 minutes 
or at 65° C. for 15 minutes kills the tubercle bacilli, provided all 
the fluid is heated to this temperature for the full period. The 
bottle should be tightly stoppered and completely immersed 
in the hot water. Dry heat at 1oo° C. for 30 minutes is effective. _ 
Against chemical disinfectants B. tuberculosis is rather resistant, — 
doubtless because of the waxy constituent of the cells. Absolute 
alcohol and mercuric chloride 1 to 500 fail to disinfect sputum 
in 24 hours. Five per cent carbolic acid is effective in this time. 
Formalin, 5 per cent solution, requires about 12 hours. B. 
tuberculosis remains alive in strong antiformin solutions (a pro- 
prietary preparation of chlorinated caustic alkali) for 30 to 60 
minutes, whereas ordinary bacteria are rapidly disintegrated by 
this chemical agent. . 

Tuberculin is a name applied to various chemical products 
of the tubercle bacillus. The oldest and most important tuber- 
culin was described by Koch in 1890. It is made by growing 
the bacillus on the surface of 4 per cent glycerin broth in shallow 
flasks at 37° C. for eight to ten weeks, steaming the cultures’ 
for one hour and filtering through porcelain, or often merely 
through paper, to remove the dead bacilli. The filtrate is then 
concentrated to one-tenth its original volume by evaporation at 
go° on the water-bath. The product keeps indefinitely in sealed 
containers and is known as Koch’s old tuberculin (‘‘alt tuber- 
kulin”). Chemical study of tuberculin has shown that the 


MYCOBACTERIACE: THE TUBERCLE BACILLUS 319 


specific active substance is a thermostable, dialyzable substance, 
insoluble in alcohol, which gives most of the protein reactions 
but not the biuret test. It is digested by pepsin and by trypsin. 
Koch’s new tuberculin, better known as tuberculin B. E. (“Bacil- 
len-emulsion’’) is made from the solid bacterial growth on glycerin 
broth. The growth is pressed between filter papers, dried and 
then pulverized in a ball mill for about three months, then sus- 
pended in 50 per cent aqueous solution of glycerin, o.oo2 gram 
of the powder to each cubic centimeter. Finally it should be 
sterilized by heating to 60° C. for 20 minutes. This tuberculin 
is a suspension, not a solution, and must be thoroughly mixed 
each time before use. Numerous other tuberculins have been 
prepared, of which perhaps the ‘Bouillon filtré” of Denys is 
the most important. It is the porcelain filtrate of the unheated’ 
glycerin-broth culture of the tubercle bacillus. It resemblesKoch’s 
old tuberculin except that it is not heated and is not concentrated. 
Inoculation of animals with B. tuberculosis gives rise to typical 
tuberculous lesions and death in most mammalian species. The 
guinea-pig is very susceptible to subcutaneous injection but 
not readily infected by the alimentary route. The lesions are 
usually well developed four or five weeks after subcutaneous inocu- 
lation and death occurs as arule in 6 to12 weeks. Rabbits are less 
susceptible to inoculation with the human type and they usually“ 
‘recover when injected with small doses of a culture, o.cor gram 
intravenously. Cattle are quite immune to this organism. Large 
doses of cultures or of sputum have been injected into calves 
and older bovines without producing tuberculosis, and quarts 
of tuberculous sputum have been fed to bovine animals with 
negative results. . 
Tuberculosis is, economically, the most important human 
disease. Approximately one death in every three between the 
age of 20 and 45, the active period of life, is due to it. It was 
recognized as a contagious disease by the ancients. Laennec,’ 


1 Léwenstein in Kolle und Wassermann, Handbuch, 1912, Bd. V, S. 554-555. 
2 For a history of tuberculosis see Landouzy: Cent ans de phtisiologie, 1808-1908, 
Sixth Internat. Cong. on Tuberculosis, Special Volume, pp. 145-189. 


320 SPECIFIC MICRO-ORGANISMS 


in 1805, by extensive post-moriem studies recognized the essential 
pathological unity of tuberculous processes. Villemin, in 1865, 
conclusively demonstrated its transmissibility by successful 
-inoculation of animals with tuberculous tissue from man and from 
cattle. 

The response of the infected tissue to the presence of the 
tubercle bacillus results in a localized mass of granulation tissue, 
the tubercle, of which the histological structure is so characteristic 
that the presence of tuberculosis may be recognized by it alone. 
From the point of introduction the bacilli may be distributed 
by the lymph or blood stream or may be carried by wandering 
cells. Eventually a bacillus comes to rest and grows slowly 
in the intercellular spaces of connective tissue. Very .soon, the 
neighboring fixed tissue elements, connective-tissue cells and 
endothelial cells, begin to multiply by karyokinesis and at the 
same time the cells become swollen with nuclei large and bladder- 
like, forming the so-called epithelioid cells. The bacilli are 
found in and between these cells. As the pathological process 
continues the nucleus of an occasional epithelioid cell divides 
many times without division of the cytoplasm, giving rise to a 
multi-nucleated giant cell. Very early in its development the 
_ peripheral portion of the tubercle becomes infiltrated with lympho- 
cytes and later, as the giant cells are formed, numerous poly- 
nuclear leukocytes are also present. Newly formed blood vessels 
are absent. With further extension, the center of the tubercle 
undergoes a caseous necrosis and liquefaction, and eventually 
this’ necrotic center enlarges so as to break through an epithelial 
surface to a passage to the exterior. This gives rise to open 
tuberculosis and tubercle bacilli may usually be found in the 
discharge from the lesion at this stage. 

The tubercle is the essential histological unit of tuberculosis. 
An infiltrated tissue may contain myriads of these tubercles in 
all stages of evolution. At any stage in its evolution the develop- 
ment of the tubercle may become arrested and it may retrogress 
and heal if the infected tissue is able to overcome the bacilli. 


MYCOBACTERIACEA: THE TUBERCLE BACILLUS 321 


If this occurs early the bacilli may be entirely destroyed and the 
abnormal tissue may disappear completely or remain only as a 
little hyaline or fibrous tissue. After caseation has occurred, 
healing results in the formation of a dense fibrous nodule, usually 
with calcareous material in the center, in which living tubercle 
bacilli can usually be demonstrated. 

The mode of infection in human tuberculosis has been a matter 
of some controversy and much of the evidence concerning it 
has been derived from animal experimentation. Unquestionably 
tubercle bacilli may pass through epithelial surfaces, especially 
of mucous membranes, without production of any demonstrable 
lesion. Ingested bacilli readily pass through the intestinal 
mucosa, especially during the digestion of fat, and they may 
first produce lesions’ in the mesenteric lymph glands, the liver 
or in the lungs. In the latter instance, they doubtless pass with 
the absorbed fat through the thoracic.duct, superior vena ‘cava 
and right heart to the pulmonary arteries. In man, the most 
important mode of infection is through inhaling the dust of dry 
powdered sputum, as a result of which lesions develop in the 
lungs. Tuberculosis may occur in any tissue of the body, reach- 
ing it through the blood and lymph. A massive infection of 
the blood stream often leads to generalized miliary tuberculosis: 
with minute tubercles in all the organs. 

The bacteriological diagnosis of the disease depends upon 
finding the tubercle bacilli in discharges from the suspected 
lesion. In sputum an acid-proof bacillus of the proper size and 
shape is almost invariably a tubercle bacillus and a diagnosis 
based upon such a finding by an experienced microscopist is 
justly regarded as very accurate. Inoculation of guinea-pigs 
will clinch the proof. The latter proceduré will also sometimes 
detect tubercle bacilli when careful microscopic search has failed. . 
In discharges from the intestine or urinary organs one may 
meet with other acid-proof organisms (B. smegmatis), and more 
care is necessary in arriving at a diagnosis. In tuberculous 


meningitis, the tubercle bacillus may be detected by. microscopic 
21 


322 SPECIFIC MICRO-ORGANISMS 


examination of the cerebrospinal fluid! in nearly every case. The 
filmy clot which usually forms in such a fluid in a half hour after 
drawing it is the most favorable material for examination. 

When a considerable amount of purulent or mucoid material 
is available for examination and one has failed to find the tubercle 
bacilli by the usual method of microscopic examination, it is 
often advisable to try some method of concentration. One of 
the common methods of general applicability is that of Uhlenhuth, 
in which antiformin is employed to dissolve the tissue elements, 
leaving the bacilli unchanged. Léffler’s modification? of the 
Uhlenbuth method is a convenient one. The material to be 
examined is mixed with an equal amount of 50 per cent anti- 
formin and brought to a boil. This dissolves the sputum or 
other material and serves to kill the bacilli. It is then cooled 
and, for each 10 c.c., 1.5 c.c. of chloroform-alcohol (z : 9) is added. 
The mixture is next violently shaken to form a fine emulsion, — 
and is then centrifugalized at high speed for 15 minutes. The. 
solid matter collects as a tough mass on top of the drop of chloro- 
form and beneath the watery liquid. This mass is rinsed in 
water, crushed between slides. mixed with a little egg albumen or 
with some of the original untreated exudate, spread, fixed, stain- 
ed and examined in the usual way. The albuminous material is 
necessary to make the preparation adhere to the slide. 

Allergic reactions are extensively employed in the diagnosis 
of tuberculosis. Tuberculin is without particular effect upon 
normal individuals but in the tuberculous individual it gives 
rise to irritation and intoxication. The phenomenon is analogous 
to that of anaphylaxis, the irritant or toxic substance being set 
free from the tuberculin by the action of specific ferments pro- 
duced and present in the body as a result of previous contact 
with the tubercle bacillus and its products. The tuberculous 


1 Holt: Amer. Journ. Dis. Children, Jan., 1911, Vol. I, pp. 26-36. Hemenway: 
ibid., 1911, Vol. I, pp. 37-41. Koplik: Johns Hopkins Hosp. Bull., 1912, Vol. XXIII, 


Pp. 113-120. 
* Williamson: Journ. A. M. A., 1912, Vol. LVIII, pp. 1005-7. 


MYCOBACTERIACEA: THE TUBERCLE BACILLUS 323 


individual is therefore sensitized to tuberculin. The sensitization 
may be local and confined to the tissue immediately surrounding 
a solitary tubercle, or it may be general as a result of more ex- 
tensive lesions. Tuberculin is applied to the skin mixed with 
an equal amount of lanolin (Moro test), or applied to a scarified 
point undiluted (Von Pirquet test), or injected into the sub- 
stance of the skin in a dose of 0.1 c.c. of 1 to 1000 dilution (Ham- 
burger intracutaneous test), or applied to the conjunctiva in a 
dose of one drop of a freshly prepared 1 per cent solution of old 
tuberculin (Wolff-Eisner or Calmette test), or finally it may be 
introduced into the circulation by subcutaneous injection of 
a dilution representing 0.00001 gram of old tuberculin, with sub- 
sequent progressive increase of the dose up to o.o10 gram if 
reaction is not obtained. The local reaction is that of irritation, 
evidenced by redness and edema, sometimes by vesiculation. 
The general reaction is evidenced by malaise, irritation at site 
of the lesion (increased cough in pulmonary tuberculosis) and 
a rise in body temperature. The reaction depends upon the 
tuberculin coming into contact with the specific ferment, and 
the location, extent and activity of the tuberculous process are 
important elements influencing the outcome of the various tests. 
Tuberculosis in the eye causes such a violent reaction to the con- 
junctival test that this method should never be employed without 
first excluding ocular tuberculosis. The subcutaneous test will 
often detect tuberculosis not revealed by the other methods. It 
is, however, a more serious procedure than the skin tests, which are 
indeed practically harmless. 

The various tuberculins are now extensively sabiovel in the 
treatment of tuberculosis, largely because of the favorable results 
obtained by Trudeau. It is given subcutaneously every 5 to 
7 days beginning first with a blank dose of salt solution and next 
with o.cooo1 gram of tuberculin. The dose is kept at the point 
at which the least general reaction possibly recognizable occurs, 
or just below this amount, the general purpose being to induce 
an immunity to tuberculin. It is often posssible to begin with a 


324 ' ‘SPECIFIC MICRO-ORGANISMS 

‘case which ‘reacts to o.coor gram of tuberculin and after treat- 
‘nent for 6 months so change the sensitiveness that 0.5 gram may be 
injected without reaction. Some cases do remarkably well when 
treated with tuberculin together with the usual: careful hygienic- 
dietetic treatment? given in sanitoria, but the value of tuberculin 
for treatment of the average case, is, perhaps, not yet fully es- 
tablished.? In general the tuberculin treatment stimulates the 
production of a thicker capsule about the healing tuberculous. 
lesion and thus tends to insure against renewed activity of the 
process at a subsequent time. 

Bacillus Tuberculosis var. Bovinus.—The bovine type of 
tubercle bacillus is found in the lesions of tuberculous cattle 
(perlsucht), frequently in hogs, in a considerable percentage of 
tuberculous lesions in children, and ‘very rarely in the tuberculous 
lungs of adult human beings. In artificial culture on solid 
media, the cell is about 1m long, shorter than that of the human 
type, and is easily stained. In glycerin broth the length of the 
cell and the staining is more irregular. On all media the growth 
is at first much less abundant than that of the human type. 
Smith has shown that the bovine type produces alkali in glycerin 
broth during the first two months, whereas the human type 
tends rather to produce acid. The virulence of the bovine 
bacillus is greater than that of the human type for all mammals, 
and it also infects birds. Intravenous injection of 0.00001 
gram of culture in thin emulsion kills rabbits with generalized 
tuberculosis in about three weeks, while a similar dose of the 
human variety is without such effect. Subcutaneous injection of 
rabbits shows a similar difference. Calves are very susceptible 
to the bovine type, not to the human. 

Tuberculosis of cattle is widely distributed and is very preva- 
lent in the older European dairy regions. The lesions are most 
common in the bronchial and retropharyngeal lymph glands, 
but they may occur anywhere in the body of the fash The 


1 Brown: Journ. A. M. A., 1912, Vol. LVIII, pp. 1678-81. 
* Brown: Amer. Journ. Med. Sciences, 1912, Vol. CXLIV, Pp. 469-624. 


MYCOBACTERIACEA: THE TUBERCLE BACILLUS 325 


disease may remain localized for years in a single lymph gland or it 
may extend rapidly causing marked emaciation and death of 
the animal. The bacilli escape from the living bovine animal 
most commonly in the feces,! sometimes in the mucus and spray 
from the nose and mouth, in the uterine dischargé and in the 
milk, and of great importance is the fact that animals may be 
excreting the bacilli without showing any gross evidence of the 
presence of the disease. Tuberculin is extensively employed in 
the detection of tuberculosis in cattle. A dose of 0.2 to 0.5 
gram diluted with 9 volumes of 0.5 per ‘cent carbolic acid is in- 
jected subcutaneously at the side of the neck. The typical 
positive reaction includes a rise in temperature of 2° or 3° F. 
over that of the previous day. The test is very accurate when 
positive but not so reliable when negative. Tuberculous animals 
should be segregated from healthy animals and food products 
from them used only after effective disinfection, or they should 
be slaughtered under inspection. 

Great interest has been manifested in the question of suscep- 
tibility of man to the bovine tubercle bacilli and the solution 
has been reached by isolating bacilli from human tissue and identi- 
fying them. Park and Krumwiede” have summarized the results 
of 1511 such examinations, and conclude that somewhat less than 
to per cent of the deaths from tuberculosis in young children 
are due to the bovine tubercle bacillus, while in adults infection 
with this bacillus is much less frequent. _ 

Bacillus Tuberculosis var. Gallinaceus (Avium).—This variety 
occurs particularly in the tuberculous lesions of barnyard fowls, 
but also in many other birds. The form of the bacillus is not 
specially characteristic except that in old cultures there is a 
marked tendency to the production of branching threads. In 
glycerin broth the growth is more delicate, and development 
takes place at the bottom of the flask as well as on the surface 


1 Briscoe and MacNeal: Ill. Agr. Exp. Sta. Bull. 149, 1911; Assn. for Tubercu- 
losis, Transactions, 1912, pp. 460-465. 
2 Journ. Med. Rsch., 1912, Vol. XXVII, pp. 109-114. 


326 ‘SPECIFIC MICRO-ORGANISMS 


of the liquid. Chickens are very susceptible to intravenous 
inoculation with this type of bacilli but quite refractory to the 
mammalian types. Mice and rabbits are also susceptible, while 
guinea-pigs are relatively resistant. The avian tubercle bacillus 
has been found in human tuberculous lesions in a very few instances. 

Bacillus Tuberculosis var. Piscium.—This variety occurs 
in natural tuberculous lesions of snakes, fish, turtles and frogs. 
The bacillus is quite different from the preceding varieties, as 
it grows rapidly on ordinary media at temperatures ranging from 
12° to 36° C., and the bacilli developed on the poorer media are 
often not at all acid-proof. When grown in bouillon with fre-. 
quent shaking the culture becomes diffusely cloudy, and the 
organisms of such cultures are said to be motile. Most warm- 
blooded animals are wholly refractory to inoculation, but, in. 
the guinea-pig, inoculation has sometimes been followed by the 
production of typical ‘tubercles with epithelioid and giant cells, 
usually encapsulated and tending to heal. 

Bacillus (Mycobacterium) Lepre.—Hansen in 1873 and 
Neisser in 1879 discovered this organism in the nodular lesions of 
leprosy. Successful artificial culture has been reported by many 
‘authors but the identity of the organisms in these cultures has 
not been established with certainty. 

B. lepre is a slender rod 0.2 to 0.454 wide by 1.5 to 6u long 
as it occurs in tissues. In its staining properties it closely re- 
sembles the tubercle bacillus. The organism occurs in enormous 
numbers in most of the nodular lesions of leprosy and is often 
abundant in the nasal mucus of these cases. When less numerous 
the antiformin method of Uhlenhuth may assist in finding them. 
For diagnosis a small piece should be excised from one of the 
nodules or fragments may be obtained from lesions in the nose or 
pharynx by means of a curette. From these pieces smears on 
slides are stained at once for acid-fast bacilli. Pieces of the 
tissue are embedded in paraffin, sectioned and stained to demon- 
strate the bacilli. 

Leprosy has been known since the dawn of history and has 


MYCOBACTERIACEZ: THE TUBERCLE BACILLUS 327 


been considered to be transmissible for a long time. It is widely 
distributed over the earth, especially in Norway, Russia, Iceland 
and in Turkey. In the United States there are leper colonies in 
Louisiana, Minnesota and in Hawaii. Lepers are occasionally seen 
in the clinics of all the larger cities. 

Leprosy is universally considered to be due to the leprosy 
bacillus, but as to mode of transmission, whether direct from 
man to man, or from the external world, or how, little or nothing 
is really known. It seems certain that the disease is always con- 
tracted in some way from a previous case, but it is certainly not 
very readily transmitted. Segregation without absolute isolation 
is the common method of handling lepers. The disease is not 
ordinarily inherited. 

Bacillus Smegmatis.—This organism occurs in the smegma 
on the genitals of man and other mammals and also in moist folds 
of the skin where there are collections of moist desquamated 
epithelium. It resembles the tubercle bacillus in form and stain- 
ing properties, but is, on the average, more readily decolorized in 
alcohol. This property cannot be relied upon to differentiate 
the two organisms in any given case. Proper care in collecting 
specimens for examination usually suffices to exclude this or- 
ganism. Urines to be examined for tubercle bacilli should be 
obtained by catheter. In doubtful cases inoculation of a guinea- 
pig is necessary. B. smegmatis has been grown in artificial culture 
and after a time adapts itself to ordinary media. 

Bacillus Moelleri—Acid-proof organisms resembling the 
tubercle bacillus in form and staining properties were found on 
timothy hay. by Moeller. The bacillus is likely to be found in 
milk and other dairy products. Probably the ‘(butter bacillus” 
of Rabinowitsch is identical with it or a near relative. When 
introduced into guinea-pigs these organisms sometimes produce 
lesions resembling tubercles. but these do not progress and kill 
the animal and a second animal inoculated from the lesions of the 
first gives a negative result. Cultures are easily obtained on 
oridinary media, and the organisms grow rapidly at 25° to 30°C. 


328 SPECIFIC MICRO-ORGANISMS - 


Other Acid-proof Organisms.—Many of the streptothrices 
which grow in the soil and upon plants are to some extent similar 
in their staining properties to the tubercle bacillus and when 
broken up into short segments may be a source of. confusion. 
These are most likely to be met with in examining agricultural 
products and especially in the feces of cattle. Mere microsccpic 
examination of such materials for tubercle bacilli has, as a rule, 
little value, as both positive and negative findings are question- 
able. Brem,! in the Canal Zone, has made the important obser- 
vation that acid-proof bacilli may grow in distilled water stored 
in bottles in the laboratory and that, when such water is used in 
preparing the microscopic objects for examination, these extrane- 
ous bacilli may be mistaken for tubercle bacilli. Burvill-Holmes? 
has made similar observations at Philadelphia. Pseudo-bacilli, 
microscopic bodies somewhat resembling tubercle bacilli, some 
times occur in microscopic preparations stained with carbol- 
fuchsin. These deceptive pictures seem to be common in prepa- 
rations of laked or digested blood.® 


1 Journ. A. M. A., 1909, Vol. LIII, pp. 909-911. 

? Proc. Path. Soc. Phila., rg10, N. S. Vol. XIII, pp. 154-160. 

3 Calmette, Sixth Internat. Cong. on Tuberculosis, 1908, Spec. Vol., p. 70; see 
also Bacmeister, Kahn and Kessler, Miinch. med. Wochenschr., Feb. 18, 1913. 


CHAPTER XXI 


BACTERIACE2: THE BACTERIA OF THE HEMOR- 
RHAGIC SEPTICEMIAS, PLAGUE AND 
MALTA FEVER 


For the bacteria of the hemorrhagic septicaemias the Com- 
mittee of the Society of American Bacteriologists has adopted the 
generic name, Pasteurella Trevisan 1887. 

Bacillus Avisepticus (Pasteurella Cholerz-gallinarum). 
Moritz! in 1869 observed this minute rod in the blood of chickens 
with chicken cholera. Toussaint (1879) and Pasteur (1880) obtain- 
ed pure cultures in liquid media and Pasteur (1880) made the far- 
reaching discovery of the method of immunization by means of at- 
tenuated bacterial cultures while working with this organism. B. 
avise pticus occurs in enormous numbersin the blood, internal organs, 
urine and feces of the acutely affected birds, in far smaller numbers 
in those having the chronic form of the disease and has also been 
found in the intestinal contents of apparently healthy birds. It 
is 0.34 wide and 0.2 to 1 in length, the shorter ones being joined 
together. It is non-motile and Gram-negative. Cultures are 
readily obtgined on ordinary media by inoculation with heart’s 
blood. Gelatin is not liquefied. Minute quantities of a virulent 
culture suffice to produce a fatal infection in chickens and many 
other birds, either by feeding or by subcutaneous injection. 
Rabbits are also extremely susceptible, guinea-pigs almost immune. 
Artificial cultures kept for three to ten months in contact with air 
are no longer capable of causing a fatal infection in chickens and 
their injection is followed by recovery and a state of immunity to 
the fully virulent organism. Acute chicken cholera is the typical 
hemorrhagic septicemia of birds, with abundant bacteria in the 
blood, and hemorrhages on the serous membranes and into the 
stomach and intestine. 

1 Vallery-Radot: Life of Pasteur, 1911, Vol. II,:p. 75. 
329 


330 SPECIFIC MICRO-ORGANISMS 


Bacillus (Pasteurella) Plurisepticus.—This name is applied to 
an organism occurring in the hemorrhagic septicemias of various 
mammals and birds. The virulence is variable and seems to be 
especially developed for the species of animal in which the organ- 
ism is foynd. It does not differ essentially from B. avisepticus. 
Other minute bacteria exhibiting the same general characteristics 
and occurring as a generalized infection in diseases of animals 
are Bacillus murisepticus in mice and Bacillus (Bacterium) rhusio- 
pathie@ suis in swine. 

Bacillus (Pasteurella) Pestis.—This organism was discovered 
simultaneously by Kitasato and Yersin in 1894 in the bodies of 


; x 
Fic. 132.—Bacillus of bubonic plague. (Yersin.) 


persons dying of bubonic plague in the epidemic at Hongkong. 
The description of Yersin has proven to be the more accurate. 
The organism is unquestionably the cause of plague, as in addi- 
tion to the evidence of animal experimentation there are several | 
instances of fatal infection of men working with the organism 
in laboratories far removed from any focus of. the disease, and 
finally the very unfortunate accident at Manila! where cholera 
vaccine mixed with a culture of B. pestis by mistake was injected 
into men and caused fatal bubonic plague. 


1 Freer: Gicsiete A. M. A., 1907, Vol. XLVIII, pp. 1264-65. 


THE’ BACTERIA OF THE HEMORRHAGIC SEPTICEMIAS 331 


B. pestis in the body of the patient is a short plump rod, 0.5 
to 0.74 wide by 1.5 to 1.84 long, and rounded at the ends. When 
stained the ends become deeply colored while the equator remains 
pale (bipolar staining). Alongside this typical form many irregu- 
lar organisms are usually found, especially longer and shorter 
bacilli, some pale, some irregularly outlined, and some swollen 
and poorly stained. The last-mentioned types of bacilli are more 
frequently found in the bodies of plague victims which have be- 
gun to decompose. They are also observed in artificial cultures. 
These irregular forms (involution forms) are important in the 
quick recognition of plague. The bacillus stains very readily, 
best with methylene blue or with a momentary exposure to carbol- 
fuchsin. Better results are obtained by fixing the spread in alco- 
hol one minute, rather than heating it. The Romanowsky: stain 
gives good results. It is distinctly Gram-negative (contrary to 
the original statement of Kitasato). Capsules may be demon- 
strated on bacilli in the peritoneal exudate of guinea-pigs and 
mice, less easily in cultures. It is non-motile and flagella have 
not been demonstrated. Spores have not been observed and 
cultures are killed at 60° C. in 10 to 4o minutes. It is also easily 
destroyed by chemical germicides, for example, by 5 per cent car- 
bolic acid in t minute. Mere drying at 35° to 37° C. kills the 
bacillus in two to three days, but at 20° C. it may withstand drying 
for 20 days. It may live for months in frozen material. 

Cultures are readily obtained on ordinary media, best at a 
temperature between 25° and 30° C. Growth is moderately 
slow. Gelatin is not liquefied. On agar containing 3 per cent 
of sodium chloride, irregular involution forms are produced in 24 
to 48 hours. Long chains are produced in broth. It does not 
form gas from sugars but does produce acid from dextrose, levu- 
lose, mannite and galactose, not from lactose or dulcite. 

The toxins of the plague bacillus are in part soluble and in 
part intimately combined with the bacterial cell. Filtrates of 
young broth cultures are without toxic properties but older broth 
cultures (14 days) yield a toxic filtrate. The bacterial cells killed 


332 SPECIFIC MICRO-ORGANISMS 


by heat produce fatal poisoning in guinea-pigs and rabbits. 
The poisons obtained so far are much less powerful than the sol- 
uble toxin of B. diphtheri@ or the endotoxins of the typhoid and 
cholera: germs. 

Rodents, especially rats and guinea-pigs, are very susceptible 

to inoculation, even a needle prick carrying the minutest quantity 
of a virulent culture being sufficient to kill in a few days. At 
autopsy the adjacent lymph nods are found greatly. swollen 
and surrounded by hemorrhagic edema. The spleen is greatly 
swollen: Everywhere are enormous numbers of the bacilli. 
Inféction by feeding gives positive results in about half the ex- 
periments. Inhalation of the bacilli produces typical pneumonic 
plague in rats. Monkeys are susceptible and present lesions 
similar to human plague. 

Bubonic plague can be recognized in descriptions of epidemics 
in very ancient records. Rufus of Ephesus who lived at the time 
of Trajan (A. D. 98) mentions specifically a very fatal acute 
bubonic plague (‘‘pestilentes bubones”). Great epidemics oc- 
curred in Europe in the 6th century (527-565 A. D.), in the four- 
teenth century (1347-1350 A. D.). Each of these was followed 
by smaller outbreaks persisting in the latter epidemic up to about 
1850. It is estimated that 25 million persons died of the plague 
in the “Great Mortality” of the r5th century. Another pandemic 
of ‘plague began in 1893. Its progress has been slow and un- 
doubtedly hampered by the prophylactic measures made possible 
by the discovery of Yersin and Kitasato. It exists as a persistent 
infection among rodents or human beings, or both, in central 
Asia, central China, northern India, Arabia, southern Egypt, 
and, more recently, seems to have established itself in California. 
Outbreaks of plague in man in new localities have usually been 
preceded or associated with mortality among rodents, especially 
rats. When an epidemic begins in a seaport town, the sewer rats 
(Mus decumanus) are first attacked. Two to three weeks later 
the house rats (Mus ratius) begin to die, and about four weeks 
later the epidemic of human plague begins. The transmission 


THE BACTERIA OF THE HEMORRHAGIC SEPTICEMIAS 333 


from animal to animal and from animal to man is accomplished 
very largely by. the agency of fleas. Rat fleas are rarely found 
on man or at large in human habitations as long as their normal 
hosts are at hand, but when the rats sicken and die of plague, 
then the fleas leave and becoming hungry they bite human beings 
and thus inoculate them with plague bacilli. 

In its permanent endemic centers, plague exists as an acute 
and chronic disease of rodents. It spreads from these regions 
through the agency of the wandering rats traveling along the 
routes of commerce and especially in ships. The infected rat, 
arrived at its destination, sets up an epizodtic among its own 
species, which later spreads to other animals and to man through 
the agency of fleas, producing the bubonic form of the disease. 
The infection may then be transmitted from man to man by 
fomites and directly by contact, and by infectious material sus- 
pended in the air, giving rise to the pneumonic form of the dis- 
ease. A persistent epizodtic of chronic plague among rodents 
in a new region may give rise to a new permanent endemic center. 

In man the disease occurs in two principal forms, the bubonic 
type, in which the portal of entry is on the skin or mucous mem- 
brane and the disease is manifested by swelling of the neighboring 
lymph nodes, and the pneumonic type in which the organisms 
are inhaled or aspirated into the lung. Both of these forms re- 
sult'in general bacteremia, as arule. The bubonic form is largely 
due to inoculation of the skin by bites of insects (fleas), while the 
pneumonic form is transmitted more directly. Other clinical types 
of the disease occur. The death rate is 30 to 90 per cent in the 
bubonic and 98 to 100 per cent in the pneumonic type. In the 
bacteriological diagnosis, the morphology of the organism in the 
tissues and in cultures, its effect upon rats and guinea-pigs, and, 
finally, agglutination of the newly isolated culture with a known 
immune pest serum are important points. 

Immunity, at least a relative immunity, follows recovery from 
the plague. Artificial immunity can be induced by injection 
of attenuated living cultures and by the injection of killed bac- 


334 SPECIFIC MICRO-ORGANISNS 


teria (Haffkine’s method). Many modifications of the latter 
are recommended and they constitute the practical method of 
vaccination against plague. Hafikine employs broth cultures 
incubated at 25 to 30° C. for six weeks under a covering of sterile 
oil. The cultures are killed at 65° C., and preserved with car- 
bolic acid. The dose is 0.1 to 0.5 c.c. for children and 3 to 4 c.c. 
for an adult man. It may be repeated after ten days. Good 
results have followed the use of this prophylactic in India. Kolle. 
suspends two-day agar cultures in broth or salt solution and kills 
at 65° C. by one to two hours exposure. Five-tenths per cent car- 
bolic acid is then added. The dose injected is the product of one 
agar culture. The vaccination should be taken by any physician 
who expects to handle plague bacilli, even if only in the laboratory. 

Horses have been immunized by Yersin, injecting first killed 
bacilli, later highly virulent bacilli, and finally the filtrates of old 
broth cultures intravenously. The serum of these horses in a 
dose of 20 c.c. confers a transient passive immunity, and has 
seemed to be of value in the treatment of a few cases of plague. 
Its preparation is so difficult and its potency so low that it has 
not come into general use. The serum has also been injected 
along with killed bacilli to confer immunity (combined active 
and passive immunization). 

The restriction and prevention of plague require measures . 
adapted to the special conditions existing. In general they include 
precautions to exclude infected animals, wholesale destruction 
of rats and other rodents and the artificial immunization of the 
human population when confronted by the disease. The eradi- 
cation of the endemic centers presents a problem of great com- 
plexity, requiring the recognition and destruction of the infected 
species of animals. 

Bacillus (Micrococcus) Melitensis.\—Bruce in 1887 dis-' 
covered this organism in the spleén of persons suffering from Malta 


1 This organism is classed as a micrococcus by most authors. It is here classed 
as a bacillus because of its general resemblance in many of its characters to B. pestis. 
None of the Gram-negative parasitic cocci resemble it in respect to physiological 
characters or in the remarkable ability to change its host. ‘ \ 


THE BACTERIA OF THE HEMORRHAGIC SEPTICEMIAS 335 


fever and obtained pure cultures. Inoculation of monkeys with 
pure cultures gives rise to a disease resembling in detail! Malta 
fever in man. 

The organism is spherical or oval, 0.3 by 0.4u in size, and is 
classed as a micrococcus by many bacteriologists. In gelatin 
cultures the cell is somewhat longer and resembles that of a true 
bacillus. The organisms are single, grouped in pairs or sometimes 
in short chains of four to five cells. Capsules and spores have 
not been observed. It is non-motile. Flagella have been de- 
tected by Gordon but other investigators have failed to confirm 
the observation. The organism stains readily and is Gram- 
negative. 

Cultures are obtained on ordinary media and growth is possi- 
ble between the extremes of 6° and 45°C. The colonies develop 
in one to three days at 37° C. and are very homogeneous. Gela- 
tin is not liquefied and neither gas nor acid is produced in media 
containing the various sugars. The organism is killed by moist 
heat at 57° C. in 10 minutes, by dry heat at g5° C. in 10 minutes 
and in 1 per cent carbolic acid in 15 minutes. It survives drying 
for several months and retains its vitality in culture without 
transplantation for several years if drying is prevented. 

Many mammals are susceptible, including guinea-pigs, rabbits, 
monkeys, rats and mice. Horses, cows, sheep and goats are not 
only susceptible to inoculation but also contract the disease natu- 
rally. In all animals the course of the infection is usually chronic 
and characterized by an irregularly remittent fever. Death is 
a common outcome in monkeys. Often the subcutaneous injec- 
tion or the feeding of a minute quantity of the culture is sufficient 
to infect, but for the smaller laboratory animals intracerebral in- 
oculation may be necessary. 

Malta fever in man is a chronic disease characterized by an 
irregularly remittent fever. Ihe spleen is enlarged and often 
the liver as well. Positive agglutination of a known culture of 
B. melitensis by the patient’s serum in dilution of 1 to 1000 is an 

1 Eyre in Kolle and Wassermann, Handbuch, rg12, Bd. IV, S. 432. 


336 SPECIFIC MICRO-ORGANISMS 


important aid in diagnosis, and isolation of the organism from the 
circulating blood, or from the spleen, and its identification makes 
the diagnosis certain. Positive cultures are more often obtained 
from the spleen, but the puncture of this organ by the inexperi- 
enced is not without danger. Blood cultures should be made dur- 
ing a febrile period and preferably late in the afternoon. Death 
occurs in 1 to 2 per cent of the cases. 

Careful investigations have shown that infection with B. meli- 
tensis is endemic among the goats of Malta, from which animals 
is obtained the milk supply of the region. The micro-organisms 
are excreted in the milk. Monkeys fed such milk acquire the 
disease, and human epidemics of Malta fever have followed the 
use of such milk under conditions closely resembling those of 
critical experimentation. Other methods of transmission have 
been tested with negative results. , 

Immunity follows recovery from the disease, but artificial 
immunization is not yet a practical success. ’ 


CHAPTER XXII 


BACTERIACE#: THE COLON, TYPHOID AND DYS- 
ENTERY BACILLI 


Bacillus Coli!.—This organism was probably observed by sev- 
eral investigators previous to 1886 but it was either neglected or 
its significance was misinterpreted. The first important study 
of it was made by Escherich in that year, who discovered it in the 
feces of healthy infants and obtained it alone on the aérobic gela- 
tin plates cultures inoculated with this material. 


Fic. 133.—Bacillus coli showing flagella. (From McFarland after Migula.) 


B. coli lives and grows in the intestinal tract of man and mam- 
mals, and organisms closely resembling it have been found in the 
intestinal canal of other vertebrates. It is discharged in large 


1 For the organisms of the colon-typhoid-dysentery group the Committee of the 
Society. of American Bacteriologists has adopted the generio name Bacterium 
Ehrenberg 1838, emended Jensen 1909. Its use in this sense may lead to con- 
fusion with the genus Bacterium Migula, especially in the minds of beginning 
students. The old generic name, Bacillus, is therefore here retained. _ 


22 337 


338 SPECIFIC MICRO-ORGANISMS 
: a 


numbers in the feces and some of these bacilli may continue their 
growth in the external world for a time. The organism is 0.4 to 
o.7 wide and 1 to 6u long, with rounded ends, usually single but 
sometimes occurring in threads. It is motile but not very active, 
and many cells, even in young cultures, may be motionless. 

There are four to eight peritrichous flagella. Spores have 
not been observed. The bacillus stains readily and is Gram- 
negative. 

Cultures develop rapidly at 37° C. on all ordinary media. 
The colony is white, opaque, often somewhat heaped up in the 


f 


Fic. 134.—Bacillus coli. Superficial colony on a gelatin plate two days old. XX 21. 
(From McFarland after Heim.) 


center and thinner near the edge. It may be round with smooth 
outline or the border may be lobulated. Under the low-power 
lens the colony appears brown, finely granular near the periphery 
and more coarsely granular near the center. It is soft and moist, 
_easily removed from the medium and easily suspended as a dif- 
fuse cloud in water. Gelatin is not liquefied. B. coli ferments 
dextrose and lactose with the production of gas as well as acid. 
It coagulates milk in 24 to 48 hours at 37° C. and renders it acid, 
produces considerable indol in pepton solution and grows abun- 
dantly on potato, often producing a brown color. 
Intraperitoneal injection of cultures into guinea-pigs and rats 


THE COLON, TYPHOID: AND DYSENTERY BACILLI 339 


causes fatal peritonitis. Subcutaneous injection may also cause 
death but frequently results in a local abscess. 

The cultures of B. coli on ordinary media are practically free 
from soluble poisons, but there is some evidence that soluble 
poisons may be produced by this organism under special condi- 
tions.!_ The bacterial cell substance is poisonous. 

As it grows in the intestine the colon bacillus is a harmless 
commensal but with a distinct tendency to invade the living 
tissue and become pathogenic whenever the normal resistance is 
lowered. The bacilli doubtless pass through the intestinal wall 
in very small numbers during absorption of the food and are de- 
stroyed in the normal body fluids and tissues in a few hours. In 
intestinal disturbances the invasive properties and the virulence 
are increased. In many other regions of the body the colon bacil- 
lus gives rise to inflammation, often purulept in character. It 
is a common cause of cystitis and pyelitis, and is an important 
agent in the causation of peritonitis following perforation of the 
intestine. Generalized infection with B. cold is rather uncommon. 
The bacilli frequently enter the blood stream from the intestine 
during the death agony, and are often present in the heart’s blood 
at autopsy, especially if this is delayed. 

The detection of B. colt in any material is ordinarily regarded 
as evidence of fecal contamination. Examinations of drinking 
water and of shell liquor from oysters are, perhaps, the most fre: 
quent applications of this principle. Fermentation tubes of 
dextrose ‘broth are inoculated with measured quantities of the 
liquid to be tested, 0.01 c.c., 0.1 c.c. and rc.c. Those cultures in 
which gas is produced are plated on litmus lactose media and the 
typical colonies transplanted to gelatin, milk, fermentation tubes 
of dextrose broth and agar slants, and for final identification the 
agglutination test with a known colon-immune serum may be 
employed. 

Bacillus (Lactis) Aerogenes.—Escherich described this organ- 
ism in 1886 as distinct from B. coli. It is non-motile, is usually 

*1See Vaughan and Novy: Cellular Toxins, Phila., 1902, p. 220. 


340 


Fic. 135.— Friedlan- 
der’s pneumobacillus; gel- 
atin stab culture, show- 
ing the typical nail-head 
appearance and the for- 
mation of gas bubbles, not 
always present. (From 
McFarland after Curtis.) 


SPECIFIC MICRO-ORGANISMS 


capsulated and its colonies are thicker and. 
less spreading. In other respects it does 
not differ materially from B. cola and many 
authorities regard it as a variety of this 
species. B. aérogenes was found by 
Escherich in the upper part of the small 
intestine. It is commonly present in or- 


-dinary cow’s milk and has been found in 


the urine in cystitis! and pyelitis. 

Bacillus (Bacterium) Pneumoniz.— 
This organism was obtained by Fried- ' 
laender in 1883 on gelatin plates inocu- 
lated with material from cases of pneu- 
monia and was confused by him with the 
organisms which he observed micro- 
scopically in abundance in his material. 
The latter were undoubtedly pneumococci 
(See Diplococcus pneumoniae p. 266). B. 
Eneumonie is rather common in the upper 
air passages and occurs in the lungs in - 
some cases of pneumonia. It isnon-motile, 
capsulated and Gram-negative, and in 
nearly all respects quite like B. aérogenes. 
The nail-shaped culture in gelatin stab is 
regarded as specially typical. 


Bacillus (Bacterium) Rhinosclero- 
matis.—This organism was described by 


von Frisch in 1882. Itis readily obtained, 
often in pure culture, by incising the lesion 
of rhinoscleroma and spreading the blood 
thus obtained on an agar surface.” It is 
also found in abundance by microscopic 

1Luetscher, Johns Hopkins Hosp. Bull., TOIT, Vol. 
XXII, pp. 361-366. 


2 Wright and Strong: New York Med. Journ., 1911, 
Vol. XCIII, pp. 516-519. 


THE COLON, TYPHOID AND DYSENTERY BACILLI 341 


examination of sections of rhinoscleruma tissue. B. rhinosclero- 
matis is capsulated, non-motile and in morphology and cultural 
characters indistinguishable from B. pneumonia. It is Gram- 
negative when stained by the usual technic. Its etiological rela- 
tion to rhinoscleroma is somewhat uncertain. 

Rhinoscleroma is a disease characterized by the occurrence of 
circumscribed grayish nodules in the mucous membrane of the 
nose, which tend slowly to extend without ulceration. Histo- 
logically the lesion is composed of granulation tissue and fibrous 
tissue with lymphocytic infiltration. Many of the cells appear 
swollen and vacuolated, so-called lace-cells, and in and near these 
the bacilli are present in large numbers. The disease occurs in 
Europe and has been seen in a number of Russian immigrants to 
the United States. 

Bacillus (Mucosus) Capsulatus and Bacillus Ozenz also occur 
on the mucous membranes of the upper air passages. They donot ap- 
pear to be specifically different from B. pneumonie of Friedlaender. 

Bacillus Enteritidis.— Gaertner in 1888 isolated this organism 
from the spleen of a man who died in an epidemic of meat poison- 
ing in which 57 persons were made ill. The meat was derived | 
from a cow, sick at the time of slaughter, and this same organism 
was found in the meat which had not been sold. The bacillus is 
of the same size and shape as B. coli, but is more actively motile 
and has more flagella. It ferments destrose with the production 
of gas, does not ferment lactose nor coagulate milk, nor does it 
produce an amount of indol appreciable by testing with sulphuric 
acid and nitrite. Its cultures are highly toxic, even after they 
have been boiled.1 A typhoid-immune serum agglutinates B. 
entertidis in fairly high dilutions. The cases of food’ poisoning 
in which it was found were characterized by vomiting and diarrhea 
and at autopsy by severe enteritis and swelling of the lymph 
follicles of the intestine. Food poisoning of this type seems to 
be rather common.? 


1 Vaughan and Novy: Cellular Toxins, 1902, p. 207. 
2 Anderson, Poisoning from Bacillus enteritidis. The Military Surgeon, 1912, 
Vol. XXXI, pp. 425-29. See also Marshall’s Microbiology, 1920. : 


, 


342 SPECIFIC MICRO-ORGANISMS 


Bacillus Suipestifer (B. Salmonii).—This organism occurs in 
the intestinal contents of hogs and in the blood in the late stages 
of hog cholera, and was for a time believed to be the cause of this 
disease. More recent studies indicate that the etiological factor 
is a filterable virus (See page 392.) B. suipestifer resembles B. 
enteritidis very closely. 

Bacillus Icteroides was described by Sanarelli in 1897 as the 
cause of yellow fever, a disease now known to be due to a different 
organism (page 374). It cannot be specifically distinguished 
from B. suipestifer. 

Bacillus Psittacosis was found by Nocard in 1892 to be the 
cause of an epidemic pneumonia transmitted to man from dis- 
eased parrots. It resembles B. coli but may be distinguished by 
inoculating parrots, for which it is extremely virulent. 

Bacillus Typhi Murium.—Léffler in 1890 found this organism 
to be the cause of a fathl epizoétic among laboratory mice. It 
forms gas and acid from dextrose, does not produce indol nor co- 
agulate milk. Mice are very susceptible and the organism has 
been employed as a practical means of destroying mice. Itseems, 
however, not to be altogether harmless for larger animals and for 
man, and it is believed that some of the paratyphoid fever fol- 
lowing food poisoning in man has been due to this particular 
organism. 

Bacillus (Fecalis) Alkaligenes.—This organism is occasionally 
found in human feces and is of importance because of the possi- 
bility of mistaking it for the typhoid bacillus, which it resembles 
in most respects. It does not produce acid from any of the sugars 
nor is it agglutinated by typhoid serum. It is not known to 
cause disease. 

Bacillus Paratyphosus.—In certain irregular fevers in man 
resembling typhoid fever there have been found bacteria somewhat 
intermediate in character between B. coli and B. typhosus. These 
have sometimes been cultured from the blood stream; at other 
times from the feces. The presence of specific agglutinins in the 
patient’s blood is further evidence of the pathogenic relationship 


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THE COLON, TYPHOID AND DYSENTERY BACILLI 345 


spread out and thinner than that of B. coli, and in semi-solid 
media the growth of B. typhosus may diffuse for quite a distance 
because of its active miotility. Dextrose is fermented with the 
production of acid but without gas. Lactose is not fermented. 
Litmus milk is rendered slightly acid and later becomes alkaline 
without coagulation. On potato the growth is almost invisible. 
In Dunham’s pepton-salt solution, iridol is not produced in suffi- 
ciently large quantities to be detected, but indol can be demon- 
strated in old cultures in 5 per cent pepton. Growth is most 
rapid at 37°-39° C., but occurs also at room temperature. 

B. typhosus is killed by moist heat at 60° C. in 10 to 15 minutes, 
and by 5 per cent carbolic acid or 1-1000 mercuric chloride in 
three to five minutes, when exposed in aqueous suspension. It 
resists drying for several days and may be alive in dry dust. The 
longevity of B. typhosus in surface waters has been studied by 
several investigators without full agreement. In general B. 
typhosus would seem to survive in such water-only for three to ten 
days except it be taken up by aquatic animals, such as the shellfish, 
when it may persist for several weeks. In soil and in frozen 
material the bacillus may live a much longer time. Freezing 
and thawing destroys a large percentage of the bacilli in a given 
liquid but does not destroy them all. 

The poisons are intimately associated with the cell substance, 

-and it is not often that culture filtrates are found to be toxic. 
The dead germ substance is somewhat poisonous, and when it 
is disintegrated by physical comminution or by digestion with 
dilute alkali at a high temperature, or by the action of serum! 
upon it, there are set free quite powerful poisons or perhaps differ- 
ent quantities of the same poison. 

The various small laboratory animals are very susceptible,to 
intraperitoneal inoculation with B. typhosus atid usually die in 
24 to 48 hours with acute peritonitis and bacteremia. The dis- 
ease produced bears no resemblance to typhoid fever in man. 
In chimpanzees a very typical attack of typhoid fever has been 

1 Zinsser: Journ. Exp. Med., 1913, Vol. XVII, pp. 117-131. 


346 SPECIFIC MICRO-ORGANISMS 


caused by feeding the organisms, with resulting lesions in the in- 
testine, bacilli.in the blood and spleen, and a continued fever. 
Typhoid fever exists generally throughout the temperate 
zone, is present throughout the year but most prevalent in the 
fall. The usual mode of infection is undoubtedly through food 
and drink. The bacilli swallowed survive in part the action of. 
the gastric juice and so gain the lumen of the duodenum. ‘The 
first multiplication seems to occur ‘here’ in a location fairly free 
from bacteria in health. The infection extends along the wall 
of the intestine, involving especially the lymphatic structures, 
solitary glands and Peyer’s patches. The bacteria pass into 
the lymph stream to be carried to the mesenteric nodes, spleen 
and into the blood. At the onset of definite symptoms of typhoid 
fever the bacilli have usually reached the general blood circu- 
lation. Subsequently the infection reaches the gall bladder, per-_ 
haps by extension along the common bile duct and cystic duct or 
perhaps by the blood stream through the liver; the organisms 
also pass through the kidney and multiply in the contents of the 
urinary bladder. They are present in the rose spots on the skin. 
The bacilli are often present in the feces in small numbers, the 
abundance of other organisms making their isolation and recog- 
nition difficult. At times localized inflammations due to B. 
typhosus develop elsewhere in the body, as in the lungs. It is 
evident therefore that the bacilli may leave the body of the patient 
through many channels, but chiefly with the urine and feces. 
Even after recdvery the patient may continue to discharge viru- 
lent bacilli for months or years. It is estimated that one per cent 
of recovered cases are presistent carriers of the infectious agent. 
The bacteriological diagnosis of typhoid fever depends upon 
isolation and recognition of the germ or detection of specific sub- 
stances in the bldod produced by the patient as a reaction to the 
presence of B. typhosus. B. typhosus is sought by blood culture 
(see page 104) diluting the blood with large amounts of broth 
(200 c.c. of broth to 2 c.c. of blood) as well as inoculating tubes 
1 Hess: Journ. Infect. Diseases, 1912, Vol. XI, pp. 71-76. 


THE COLON, TYPHOID AND DYSENTERY BACILLI 347 


of bile and the usual agar plates; by cultures from the rose spots, 
and by cultures inoculated with duodenal fluid. These methods 
are likely to be successful very early in the disease. Later it is 
well to make cultural examination of the feces and urine, especially 
just before discharging a recovered patient. 

The detection of B. typhosus in feces requires special care. It 
is essential that the primary plate cultures should be made upon 
some medium which will specially favor the detection of colonies 

‘of this organism. Several different technical methods have been 
developed of which the most important are those of Krumwiede 
and his coworkers and of Russell and his followers at the Army 
Medical School. Krumwiede! and Pratt employ an agar contain- 
ing brilliant green, which exercises a relative inhibition upon most 
of the other fecal bacteria, thus favoring the development and 
detection of B. typhosus. Russell’s method as employed in the 
U.S. Army utilizes Endo’s medium.? The fecal material is mixed 
with broth to form an opaque suspension. A drop of this sus- 
pension is transferred to the surface of an Endo plate and is spread 
over the surface by means of a sterile bent glass rod. After in- 
cubation at 37° C. for 24 hours the typhoid, paratyphoid and dys- 
entery bacilli appear as small, clear, almost colorless, transparent 


1 Krumwiede, Pratt and McWilliams, Journ. Infectious Diseases, 1916, 18, 1. 

? For Endo’s medium a stiff lactose agar is prepared containing Liebig’s extract 
5 grams, salt 5 grams, pepton 10 grams, lactose ro grams and agar 30 grams in 1000 
c.c: of water. This is sterilized in flasks containing 100 c.c. each. When needed 
the contents of a flask are liquefied, enough sodium hydroxide is added to make the 
reaction 0.2 per cent acid to phenolphthalein and to it are then added 10 drops of 
saturated alcoholic solution of basic fuchsin, and approximately 20 drops of a freshly 
prepared solution of sodium sulphite, or just sufficient to decolorize the fuchsin 
The amounts of fuchsin and of sulphite may be altered to suit different lots of 
medium and it is well to test out several different quantities upon known cultures 
of typhoid, paratyphoid and various dysentery bacilli before deciding upon the 
exact amounts of fuchsin and of sulphite of particular sampleg to be used. Both 
of these substances, but especially the sulphite, are subject to variation in com- 
position as they are obtained in the market. The material is well mixed and poured 
into 8 or 10 Petri dishes, allowed to solidify and dried in the incubator to remove 
water from the surface before use. Large Petri dishes, 15 cm. in diameter, are 
preferable. . 


348 SPECIFIC MICRO-ORGANISMS 


colonies while the colov and aerogenes bacilli will have produced 
larger colonies colored pink tc deep red or even showing a metallic 
_ luster of the fuchsin. In every test it is wise to inoculate one plate 
with a fecal suspension to which a known culture of the organism 
in question has been added so that there may be known standard 
colonies for comparison. The suspected colonies are then, trans- 
planted to Russell’s double-sugar medium, an agar! containing 
1 per cent lactose, o.1 per cent glucose and Andrade’s indicator. 
This medium is slanted so that the lower end of the tube is entirely 
filled with agar for a depth of one half inch with the inclined sur- 
face above this. Each colony is transplanted by stabbing the 
inoculating needle deep into the cylindrical butt of the tube and 
also stroking the inclined surface, the result being a combined 
streak and stab-culture. After incubation for 24 hours any of 
these cultures which show a growth characteristic of the typhoid 
bacillus, that is, pink butt and almost colorless upper portion 
under the streak, without any gas bubbles, are tested for aggluti- 
nation against a known anti-typhoid agglutinating serum. The 
examination is thus completed in two or three days. 

The specific antibody ordinarly sought in the blood is the 
typhoid agglutinin. A few drops of blood in a Wright’s capsule 
suffice for the microscopic test (see page 218). A young active 
culture (breth three hours) of 2 known B. typhosus is used, and 
the serum is tested in dilutions of 1:20, 1:40 and 1:80, observed 
for an hour. Normal serum rarely shows any clumping in any of 
these dilutions at the end of an hour. This agglutination test is 
of little or no value if the patient has received typhoid vaccine 
within a year. 

Dreyer? and his coworkers have devised a technic for measuring 


1 The double-sugar medium is a 2 to 3 per cent agar, neutral to litmus, to which 
has been added 1 per cent lactose and 0.1 per cent glucose. On this medium B. 
typhosus does not change the color when it is growing on the surface, but produces 
a red (acid) color about the stab. See Russell, Journ. Med. Rsch., 1911, Vol. XX, 
Pp. 217-229. ’ 

* Dreyer and Inman, Lancet, July 31, 1915, p. 225; Dreyer and Torrens, ibid., 
1915, ii, p. 1369; Dreyer, Walker and Gibson, ibid., Feb. 13, 1915, p. 324; Dreyer and 


THE COLON, TYPHOID AND DYSENTERY BACILLI 349 , 


the agglutination titre of a serum more accurately than has pre- 
viously keen possible. A standard agglutinable culture is em- 
ployed. This is a formolized suspension of B. typhosus prepared:so 
as to possess a standard opacity and a definite agglutinability. 
By this method it has been possible to demonstrate a distinct in- 
crease in agglutinins from the sixteenth to the twenty-fourth 
day of typhoid fever. Professor Dreyer and his followers have 
employed this test to diagnose enteric fevers (typhoid and para- 
typhoids) in patients who had been previously inoculated with 
the corresponding bacterial vaccines. Pappenheimer! has dem- 
onstrated conclusively, however, that such a rise in agglutinins 
in inoculated men is not a reliable criterion for diagnosis. In 
such cases the diagnosis of typhoid fever can be proven only by 
detection of the typhoid bacillus, or by the demonstration of the 
typical pathological changes at autopsy. 

Transmission of the disease takes place in a variety of ways. 
To the best of our knowledge, the typhoid bacilli come only from 
human individuals infected with them. Some of these actually 
suffer from typhoid fever, while others are merely healthy carriers 
of the infection. From them as centers the bacilli are distributed 
by contact and by intermediate object. B. typhosus is able to | 
live for a considerable time in the external world, probably for 
one to three weeks in ordinary surface waters and longer in soil. 
It is able to grow and multiply in some foods, especially milk. 
Water supplies contaminated with feces and urine from patients 
or from healthy carriers have unquestionably been an important 
factor in the causation of typhoid fever in the past, and the pro- 
vision of a supply of drinking water free from all suspicion of 
recent mixture with sewage is the first step in the control of this 


Walker, ibid., Sept. 2, 1916, p. 419; Walker, ibid., Nov. 25, 1916, p. 896; Dreyer, 
Gibson and Walker, ibid., Apr. 8, 1917, p. 766; Walker, zbid., Vol. I, p.17. Walker, 
ibid., April 14, 1917, p. 568; Dreyer and Inman, ibid., March 20, 1917, p. 365. 
Fennel, Journ. Amer. Med. Assn., 1918, 70, p. 590. 

1 Pappenheimer, Trench Fever—Report of Commission, Medical Research 
Committee, American Red Cross, Oxford Press, 1918, pp. 80-142. 


350 SPECIFIC MICRO-ORGANISMS . 


disease in a community. The infected oyster from a sewage. 
polluted oyster bed is another source of typhoid fever. The 
contamination of food by permanent carriers of the bacilli is 
difficult to control. All possible means need to be employed. tc 
prevent these persons from handling foods prepared for consump- 
tion, and especially milk. Flies (Musca domestica) are important 
aids in the transfer of bacilli from discharges containing them, 
especially from open privies, to. foods exposed for sale or being 
prepared in neighboring unscreened kitchens. 

The prevention of typhoid fever by restricting the distribu- 
tion of the bacilli bas been only partially successful in civil life 
and in armies on a war footing it has proven wholly ineffective. 
Vaccination to prevent typhoid fever was first extensively prac- 
tised by Wright in the British army. Russell,! following the 
method developed by Wright and Leishman, has prepared a vaccine 
with which practically the whole U. S. army has been inoculated, 
The vaccine is a suspension of B. typhosus in salt solution, stand- 
ardized by microscopic count of the bacterial cells, sterilized by 
heating at 53° to 56° for an hour and preserved by the addition 
of 0.25 per cent trikersol. Three injections are given subcutane- 
ously at intervals of 10 days, 500 million bacilli at the first dose 
and 1doo million at each of the following doses. The results 
in the U. S. army have been remarkably good, rivaling those 
obtained with the use of. vaccinia in the prevention of small- 
pox. 

Experience has shown, however, that the immunity conferred 
in this way is not always adequate to prevent the occurrence 
of typhoid fever. A considerable number, in the aggregate, of 
cases of bacteriologically proved typhoid fever occurred in the 
American Expeditionary Forces in France in 1918 and rgrg and 
in a few instances definite outbreaks of the disease occurred, such 
that they might be termed small epidemics. Nevertheless it 
may justly be concluded that typhoid vaccination has relegated 


Russell: Boston Med. and Surg. Journ., 1911, Vol. CLXIV, pp. 1-8; Harvey 
Lecture, 1913. as 


THE COLON, TYPHOID AND DYSENTERY BACILLI 351 


typhoid fever to a very minor position as a cause of illness and 
death in armies, whereas in previous wars it has often shown itself 
the chief cause of death. 

Bacillus (Bacterium) Dysenteriz.—Shiga in 1898 isolated 
this organism from the feces of patients suffering from dysentery, 
showed that it is agglutinated by the blood of dysenteric patients 
in high dilutions and not by normal human blood. 

B. dysenterie is about 0.6 in width by 2 to 4 in length, usually 
single and non-motile. It stains readily and is Gram-negative. 
Involution forms are common in older cultures. The organism 
grows readily on ordinary media and its cultures resemble those 
of B. typhosus very closely. Gelatin is not liquefied; no indol is 
produced in pepton solution; no gas is formed from any of the 
sugars; milk is rendered slightly acid and then alkaline without 
coagulation. It differs from the typhoid bacillus in failing to 
ferment mannite and maltose. 

When cultures are injected intravenously into rabbits severe 
diarrhea is produced, which may be bloody. The animal usually 
dies in a few days, and if it recovers often exhibits paralysis of 
the hind legs.. Similar results are obtained by the injection of 
dead bacilli, indicating that the effect is toxic rather than infec- 
tious. Kittens and puppies have been infected by introducing 
dysentery bacilli into the stomach, resulting in diarrhea with the 
intestinal lesions of dysentery. The toxins seem to be intimately 
bound up in the cells in young cultures, but readily set free 
into solution after the bacilli are killed. Culture filtrates, of 
which 0.02 c.c. suffices to kill a rabbit in 24 hours, have been 
obtained. 

Acute epidemic dysentery is the disease in which this organism 
isfound. The infectious agent is found on the membrane of the 
large intestine, which is diffusely inflamed, often covered with a 
fibrinous exudate, or by a pseudo-membrane. Later numerous 
ulcers may be formed. The bacillus is also present in the feces, 
especially during the first few days of the disease. It may be 
found by plating on Endo’s medium by the same method as has 


352 SPECIFIC MICRO-ORGANISMS 


been described for typhoid bacilli. After the first week of the 
disease, search for the bacillus is less promising. The bacilli 
are only very rarely found in the blood or internal organs. The 
blood of the patient agglutinates the bacillus of Shiga in dilutions 
of 1 to 50 or 1 to 100. The mortality is about 25 per cent, but 
variable in different epidemics. 

Horses have been immunized with cultures of B. dysenterie and 
the serum of these animals has been found to be antitoxic as well as 
bactericidal. Its use in treatment has given promising results 
and seems to cause a reduction in the death rate of about 50 per 
cent. 

Paradysentery Bacilli—Flexner in 1899: isolated a badilus 
from cases of dysentery in the Philippines which at the time was 
considered to be the same as the Shiga bacillus. Kruse, although 
he found the Shiga bacillus in epidemic dysentery, found a some- 


what different organism in “asylum dysentery” or pseudo-, 
dysentery, which proved to be identical with the Flexner bacillus. — 


Between 1901 and 1903 a number of strains of bacilli resembling 


somewhat B. dysenteri@ were isolated by different investigators © 


from epidemics of diarrheal disorder, especially in the Eastern 
United States. The paradysentery bacilli are indistinguishable 
from B. dysenterie in morphology or in cultures on ordinary 
media. They are all much less toxic to rabbits than the. Shiga 
bacillus, and they all ferment mannite with the production of 
acid, while the Shiga bacillus does not. 

The bacteria considered in this chapter are all inhabitants 
of the alimentary canal (mouth, pharynx, intestine) of man or 
other mammals. They are small bacilli, Gram-negative, without 
spores and without the ability to liquefy gelatin. They vary 
from each other in motility, possession of flagella, possession of 
capsules, and in their ability to form poisonous substances and 
to ferment various carbohydrates. Media containing various 
carbohydrates along with an indicator such as litmus to show 
the production of acid, and contained in fermentation tubes so 
as to measure the production of gas, are very useful in differentiat- 


% 


THE COLON, TYPHOID AND DYSENTERY BACILLI 353 


ing! the various types of bacteria in this group. Thus, in a 
broth containing maltose, B. typhosus produces acid, B.coli 
produces acid and gas, and B. dysenterie produces neither. 
Specific agglutination with the serum of an animal immunized 
with a known culture constitutes the most important test in the 
identification of unknown forms falling within this group. This 
test may be used with the capsulated- species after they have 
lost the tendency to form capsules through propagation on artifi- 
cial media.” For a detailed discussion of the classification and 
fermentative reactions of the colon-typhoid group reference may 
be had to the paper of Winslow,’ Kligler and Rothberg, with 
which is included an extensive bibliograpky. 


1 Hiss has devised a very useful medium for this purpose which obviates the neces- 
sity of using the fermentation tube to detect the gas. His serum-water medium is 
made by mixing beef serum, 1 part, with distilled water, 2 to 3 parts, and steaming 
15 minutes to destroy enzymes. , Pure litmus solution (about 1 part of a 5 per cent 
solution to roo parts of the medium) is then added to preduce a deep blue color. 
The medium is divided into several portions and x per cent of the desired carbo- 
hydrate is added to its respective portion. The sugar serum-water media are then 
sterilized at 100° C., on three days. Fermentation is shown not only by the redden- 
ing of the litmus but also by coagulation of the liquid medium, and gas production 
is shown by bubbles caught in the coagulum. (Hiss and Zinsser: Text-book of 
Bacteriology, 1910, p. 132.) 

2 Fitzgerald: Proc. Soc. Biol. and Med., 1913, Vol. X, pp. 52-53. 

3 Winslow, Kligler and Rothberg, Journal of Bacteriology, 1919, 4, 429-503. 


23 


- CHAPTER XXIII 


BACTERIACEH: BACILLUS MALLEI AND MISCELLA- 
NEOUS BACILLI 


Bacillus (Bacterium) Mallei—Léffler and Schiitz in 1882 
obtained pure cultures of this organism from glandered horses. 
and produced glanders by the injection of these pure cultures. 

The bacillus is 0.3 to 0.5» wide and 2 to 5y long, usually 
straight with rounded ends, but sometimes irregular in shape. 
Filamentous and branched forms have been observed in cultures. 


Fic. 139.—Bacillus mallei from an agar culture. xX 1100. (After Park and 
‘Williams.) 


It is not motile. Spores have not been observed. B. mallet is 
stained with moderate difficulty and often stains unevenly like 
the tubercle and diphtheria bacilli. After being stained, the 
bacterium is easily decolorized in weak acid or alcohol; it is also 
Gram-negative. Cultures develop on ordinary media, better on 
glycerinated media, at temperatures ranging from 22° to 42° C., 
best at 37° C. On Potato at 37° C. a viscid yellowish-brown 
354 


BACILLUS MALLEI AND MISCELLANEOUS BACILLI 355 ; 


growth develops surrounded by a greenish stain on the potato. 
Gelatin is not liquefied. The organism is killed by moist heat 
at 55° C. in 10 minutes, and in-2 to 5 minutes by 5 per cent car- 
bolic acid or‘1 to rooo mercuric chloride. 1t survives drying 
for only a few weeks and dies out quickly~in water. , Many 
mammals are susceptible to inoculation, including horses, guinea- 
pigs, cats and dogs. Cattle.are immune. Man is susceptible 
and human glanders frequently ends in death. 

Mallein is analogous to tuberculin. A culture in glycerin 
broth incubated for six weeks is steamed and filtered, and the 
filtrate evaporated to one-tenth the original volume is the mallein. 
This substance is toxic to animals suffering from glanders but 
not poisonous to healthy animals. 

Glanders is a disease most common in horses, mules and asses. 
It begins as an inflammation of the nasal mucosa with localized 
‘nodular infiltrations which later ulcerate. The infection may 
become generalized at once causing acute glanders and death in 
one to six weeks, or it may progress very slowly and persist for 
years as chronic glanders. The chronic type is common in horses. 
After apparent recovery from the disease, nodules containing 
living bacilli may be found in the lungs. Histologically the gland- 
ers nodule consists of granulation tissue infiltrated with leukocytes 
and tending to become purulent at the center. The bacilli leave 
the body in the nasal secretion and in the discharge from ulcers. 
Infection of equines takes place most frequently by ingestion of 
food soiled by these discharges. In man the disease seems to 
result from inoculation of small wounds in the skin. It often 
runs an acute course terminating in death, but chronic glanders 
with recovery also occurs in man. A few sad laboratory accidents 
in which workers have become inoculated with glanders have 
emphasized the necessity for caution in handling this organism. 

The bacteriological diagnosis depends upon (1) identification 
of B. mallei, (2) reaction of the animal to mallein, (3) agglutina- 
tion reaction, and (4) complement fixation. For the recognition 
of the bacillus, some of the suspected material is suspended in 


356 SPECIFIC MICRO-ORGANISMS 


broth and injected into the peritoneal cavity of a male guinea-pig 
(method of Straus). If B. mallei is present a general inflamma- 
tion of the peritoneum develops and after three or four days the 
testicles of the animal become swollen, inflamed and later suppu- 
rate. They may burst through the scrotum. Cultures should 
be made from this pus on plates of glycerin agar and the colonies © 
transplanted to potato at 37° C. Very few other organisms 
give rise to a similar pathological picture in the guinea-pig. At 
the same time the mallein test is carried out by injecting 0.2 c.c. 
of the concentrated mallein diluted with 0.25 per cent solution: 
of carbolic acid into the suspected horse. The presence of gland- 
ers is indicated by a rise in temperature of 2° to 5° F., signs of 
general intoxication, and especially by swelling and inflammation 
at the site of injection. For the agglutination test the serum 
is diluted to 1:500 to 1:3000. Positive results with lower dilu- 
tions may apparently be given by normal horses. The comple- 
ment-fixation test follows the principles of Wassermann test for | 
syphilis, a culture of B. mallei being employed as antigen.’ At- — 
tempts at immunization have not been practically successful. 
Bacillus (Bacterium) Abortus.—Bang and Stribolt, working 
in Denmark in 1897, isolated this organism from the uterus of a 
cow suffering from the disease known as contagious abortion, 
and reproduced the disease by inoculating healthy cows with 
these cultures, The same organism was isolated by MacNeal 
and Kerr? in 1910 from aborting cows in the United States. The 
organism is of interest because of its behavior toward oxygen when 
first isolated. It fails to grow in the air or in hydrogen, but growsin 
a partial pressure of oxygen somewhat below that of the atmos- 
phere. The bacillus is pathogenic for a number of different 
mammals, and in guinea-pigs it causes granulomatous lesions 
resembling somewhat those of tuberculosis.? The organism - 


1 Mohler and Fichorn: Twenty-seventh Annual Rep. Bur. Anim. Industry, U. 5. 
Dept. Agr., 1910; reprinted as Circular 191 (1912). ; 

? MacNeal and Kerr, Journ. Infectious Diseases, 1910, 7, p. 469. 

3Smith and Fabyan: Cenir. f. Bakt., I, Abt. Orig., 1912, Bd. LXI, S. 549-555. 
Fabyan, Journ. Med. Rsch., 1912, Vol. XXV, p. 441-488. 


BACILLUS MALLET AND MISCELLANEOUS BACILLI 357 


occurs rather frequently in market milk. It is not known to infect 
man. 

Bacillus (Bacterium) Acne.—This minute non-motile organ- 
ism, first described by Gilchrist, is constantly present in the pap- 
. ules and pustules of the common skin affection, acne vulgaris. 
Cultures are most readily obtained by expressing, with careful 
asepsis, some of the cheesy pus from a recent papule and mixing 
it with 2 c.c. of ascitic fluid in a test-tube. Dilutions from this 
are made to similar amounts of ascitic fluid in series (about five 
tubes in all). To the first tube one adds 8 c.c. of sterile glucose 
broth and covers this with a layer of paraffin oil (albolene). Toeach 
of the remaining tubes are then added 8 c.c. of melted glucose 
agar cooled to 50° C., the contents of each tube mixed without 
introducing air bubbles and then quickly solidified by immersion 
in cold water. The colonies of B. acne develop at 37° C. after 
' five to ten days, beginning about 8 mm. beneath the surface, and 

they grow best in a narrow zone about 5 mm. in depth. The 
colonies attain a large size (3 mm.) and an abundant supply 
of bacillary substance for preparation of vaccine may be obtained 
by thrusting a sterile glass capillary into such a colony. In 
its behavior to oxygen when first isolated the organism exhibits - 
the same peculiarity as the bacillus mentioned in the preceding 
paragraph. 

Sometimes the agar cultures fail. In that event one may 
.Tepeat the series of ascitic-glucose-agar dilution cultures by 
inoculating with sediment from the ascitic-broth tube, which has 
been incubated ten days. This broth culture usually develops 
an abundant: growth of staphylococci for the first few days but 
after ten days the cocci will have disintegrated to a considerable 
extent and B. acne will usually have become the most numerous 
organism in the sediment. 

Bacillus Fusiformis (Vincenti).—In an ulcerative disease of 
the tonsils, known as Vincent’s angina there occur very large 
numbers of fusiform rods, 0.3 to 0.8u in thickness and 3 to rou long, 
associated with spiral filaments with rather coarse windings. 


358 SPECIFIC MICRO-ORGANISMS 


These associated organisms also occur in other ulcerative con- 
ditions of the mouth and pharynx and rarely elsewhere in the body. 
The spiral filaments are evidently ordinary mouth spirochetes. 
The fusiform bacillus is an anaerobe and it has been grown in 
artificial culture.? 

Bacillus (Lactobacillus) Bifidus.— Tissier in 1898 showed that 
the Gram-positive bacillus predominant in the stools of healthy 
nurslings is not a form of B. coli as had been supposed since the 
investigations of Escherich (1886) but is an entirely different 
organism. He obtained cultures by making a series of dilutions 
(five to ten tubes) in tall tubes of glucose agar by the method of ' 
Veillon (see page 116). The colonies develop best about 1 to 2 
cm beneath the surface after three to eight days at 37° C. In 
these colonies many of the bacilli show dichotomous branching. 
Bifid forms are also sometimes seen in stools and in mixed cul- 
tures in broth. The organism produces a strong acid reaction 
and the cultures soon die out. The bifid forms are doubtless 
involutions due to presence of unfavorable amounts of acid. 

Bacillus (Lactobacillus) Bulgaricus.— This organism isa rather 
large rod 1 by 6 approximately. It occurs in milk and milk 
products and is especially abundant in milk fermented at 40° C. 
for three or four days. Colonies may be obtained on plates of 
milk agar (1:2) incubated at 37° C. in hydrogen. A high degree 
of acidity (lactic acid) is produced in the cultures of this organism, 
and it is employed to some extent in the Prepanabionl of acid-milk- 
beverages. 

Bacillus (Proteus) Vulgaris—Hauser in 1885 discovered 
this organism in putrefying infusions of animal matter. It is an 
actively motile rod 0.6 in thickness and exceedingly variable in 
length, with abundant flagella. Spores have not been observed. 
It is universally distributed in the soil and is abundant in putrefy- 
ing flesh. Gelatin is rapidly liquefied. Food poisoning in man 
has been ascribed to this organism. It is also capable of infecting 
laboratory animals when injected in large doses. 


1 Tunnicliff: Journ. Infectious Diseases, 1906, 3, p. 148; ibid., 1912, 10, p. I. 


BACILLUS MALLEI AND MISCELLANEOUS BACILLI 359 


Bacillus Pyocyaneus (Pseudomonas Pyocyanea).—Gessard 
in 1882 isolated this organism from green pus. It is a slender 
rod, actively motile. A soluble blue-green pigment is produced 

‘in the cultures. Gelatin is liquefied. Guinea-pigs are susceptible 
to intraperitoneal inoculation. In man the organism is most 
common in the pus from wounds, where its presence is considered 
as only mildy deleterious. The bacillus has also been found in 
otitis media and a few cases of fatal generalized infection with B. 
pyocyaneus have been described. 

Bacillus Fluorescens var. Putidus.—This non-pathogenic 
actively motile rod is common in putrefying material. It pro- 

_ duces spores when grown on quince jelly. The greenish-yellow 
pigment is soluble in water. Gelatin is not liquefied. A number 
of different fluorescing bacilli have been found in the soil and 
surface waters. Some of them liquefy gelatin. 

Bacillus Violaceus.—This is a non-pathogenic water bacterium 
which produces a pigment of deep violet color. It is actively 
motile and liquefies gelatin rapidly. The pigment is not soluble 
in water. Several different bacteria are known which produce 
a violet pigment. 

Bacillus Cyanogenus (Pseudomonas Syncyanea).—This non- 
pathogenic actively motile organism produces a_bluish-black 

~pigment which is soluble in water. Gelatin is not liquefied. 
B. cyanogenus sometimes causes trouble in dairies as its growth 
in milk imparts a blue color to it. 7 

Bacillus Prodigiosus.—This small oval organism grows rapidly 
at room temperature on ordinary media, and is occasionally 
observed on foodstuffs such as moist bread and potatoes. Ordi- 
narily it is encapsulated and non-motile, but it sometimes possesses 
flagella. Gelatin is rapidly liquefied. A red pigment is produced 
at room temperature but not at 37° C. This pigment is insoluble 
in water. Large doses of B. prodigiosus injected into animals 
sometimes give rise to signs of intoxication. 


CHAPTER XXIV 
SPIRILLACEZ AND THE DISEASES CAUSED BY THEM 


Spirillum Rubrum.—Esmarch discovered this organism in 
the body of a dead mouse. It is of chief interest as a harmless 
example of spiral bacterium for class study. It grows rather 
slowly at room temperature without liquefying gelatin. A dull 
red pigment, insoluble in water, is produced even in the absence 
of oxygen. Growth occurs at 37° and also in the refrigerator at 
5° to 10° C. When grown at temperatures above 20° C. the 
organism is a relatively short, slightly bent rod‘and its spiral 
nature is not very evident. At 10° C. beautiful long spirals: are 
produced in broth cultures. It is actively motile. 

Spirillum Cholere (Vibrio Cholerze).— Koch in 1883 discovered 
this organism in the intestinal discharges of patients suffering 
from Asiatic cholera, and continuing his studies in India in the 
same year established this organism as the probable cause of 
cholera. It occurs in the intestinal contents and feces of cholera 
patients, often in great abundance, rarely in the feces of healthy 
persons, and has been found at times in surface waters, and in 
drinking water during epidemics of cholera. 

Sp. cholere is a curved cylinder about o.4y in thickness and 
1.54in length. In older cultures in broth long spiral forms occur. 
There is considerable variation in shape in cultures older than 
48 hours. The organism is actively motile and possesses a single 
flagellum at one end. Those short spirals showing more than 
one flagellum are not to be regarded as true cholera germs. Spores 
have not been observed. The spirillum stains readily and is 
Gram-negative. 

It grows well and rapidly on ordinary media. The reaction 
needs to be distinctly alkaline to litmys as the organism is very 


260 


SPIRILLACEZ AND THE DISEASES CAUSED BY THEM 361 


sensitive to acids. Colonies appear on gelatin at 22° C. in about 
24 hours as circular disks with somewhat irregular border and 
granular interior. A few hours, later the gelatin begins to liquefy. 
In pepton-salt solution both indol and nitrate are formed, so that 
the addition of sulphuric acid gives rise to the red color due to 
nitroso-indol. This has been called the cholera-red reaction, but 
it is of course not a specific test for this organism. In milk there 
occurs abundant growth without apparent change in the medium. 
In broth, growth is extremely rapid and a pellicle forms in 24 


Fic. 140.—Cholera vibrios, short forms. (From Kolle and Schiirmann after Zetinow. 


hours. The rapid growth in pepton solution (pepton 1 per cent, 
salt o.5 per cent) and the tendency for the organisms to collect : 
at the surface are utilized in practical enrichment for purposes 
of diagnosis. The spirillum is an obligate aérobe. It is very 
easily killed. If dried on a cover-glass at 37° C., the organisms 
are all dead in two hours. It seems impossible, therefore, for the 
infection to be distributed in dry dust. Moist heat at 56° C. 
kills the cholera spirilla in 30 minutes. They are also easily 
killed by chemical germicides. Milk of lime is recommended for 
the disinfection of excreta. The organism lives for several weeks 


SOF 


362 SPECIFIC MICRO-ORGANISMS 


in surface waters but certain waters, as for example the Ganges 
River, destroy the cholera spirilla very quickly. This property 
has been ascribed to a weak acidity of the water. 


Fic. 141.—Cholera vibrios, longer forms at higher magnification, showing long 
flagella. (From Kolle and Schiirmann after Zetinow.) 


Animals are not naturally susceptible to cholera. “Koch gave 
to a guinea-pig 5 c.c. of a 5 per cent solution of sodium carbonate 


isis ee ~~ ) cae 
in} 

épmo a eo: 

fe y*) ~eas 
© @‘< 
ac i 


Fic.. 142.—Involution forms of the spirillum of cholera. (Van Ermengem.) . 


through a tube, and then 5 to 10 c.c. of water containing cholera 
spirilla. The animal then received 1 c.c. of tincture of opium 


SPIRILLACEA AND THE DISEASES CAUSED BY THEM 363 


per 200 grams of body weight, injected into the peritoneal cavity. 
In this way a condition resembling cholera in man was induced, 
and the animals died in 24 to 36 hours. Autopsy revealed severe 
enteritis, and abundant cholera spirilla in the intestine. Similar 
results may be obtained, however, when other organisms. are 
substituted for the cholera germs in this procedure. Intravenous 
injection of cultures into rabbits, and feeding of virulent cultures 
to very young rabbits gives rise to rather typical cholera in many’ 
of the animals. Intraperitoneal injection of cultures into guinea- 
pigs gives rise to fatal peritonitis. Pigeons are relatively immune. 

The poisons of the cholera germ are intimately connected 
with the substance of the living cell. Culture filtrates are slightly 
or not at all poisonous. The dead bacterial cells are poisonous, 
but the poison in them is a very labile substance and readily altered 
by heat. It seems to become soluble when the cell disintegrates, 
and this may explain the poisonous properties sometimes observed 
in the filtrates of older cultures. 

Immunity to this organism was obtained by Pfeiffer by inject- 
ing non-fatal doses into guinea-pigs. When a small amount of 
culture is injected into the peritoneal cavity of such an immune 
animal, the bacteria become quickly clumped together and are 
then rapidly disintegrated and dissolved in the peritoneal fluid. 
This is known as Pfeiffer’s phenomenon and was the first example 
of cytolysis to be observed. The solution of the bacteria sets 
free their poison and if a very large dose has been injected the 
animal may be killed by this poison regardless of his immunity to 
the living germs. 

Asiatic cholera seems to have existed in India for many 
centuries and there are reliable records of its occurrence there 
in the sixteenth, seventeenth and eighteenth centuries. The 
first recognized great world invasion of cholera began in 1817 
and ended in 1823. Succeeding pandemics occurred in 1826- 
1837, 1846-1862, and 1864-1875. The fifth invasion began 
in 1883 and ended shortly after the great outbreak at Hamburg 
in 1892. The sixth epidemic began in 1902 and has involved 


364 SPECIFIC MICRO-ORGANISMS 


Egypt; Russia, Turkey and Italy. The fifth and sixth invasions 
have been very much restricted, largely without doubt because 
of the modern methods founded upon knowledge of its causation. 
Cholera was epidemic in the United States in 1833-35, 1848-54, 
1871-73, and there were a few cases in 1893 and again in 1910. 
This disease occurs as a protracted epidemic in which the infection. 
passes from person to person, and as an explosive epidemic in 
which many people are stricken at once as a result of contamination 
of the public water-supply. 

The causal relationship of Spirillum cholere to human Asiatic 
cholera is no longer questioned. Several laboratory workers 
among them R. Pfeiffer and E. Oergel, have suffered typical 
attacks of the disease as a result of accidental laboratory inoculation. 
Dr. Oergel received some peritoneal fluid from an inoculated guinea- 
pig into his mouth and he died of cholera. Pettenkoffer and 
Emmerich, in order to disprove the supposed causal relation of 
this organism to cholera, took some alkaline water and then water 
containing a minute quantity of a fresh culture. The former 
investigator had a severe diarrhea and the latter a severe and 
dangerous attack of typical cholera from which he eventually re- 
covered. The organism was recovered from the stools in all these 
instances. 

The cholera spirilla enter the body with the food and drink 
and if they escape the germicidal action of the gastric juice they 
may establish themselves in the intestine. In an acute case of 
cholera they multiply here enormously and induce a severe 
enteritis in which large quantities of fluid are secreted into the 
lumen of the intestine and discharged from the rectum along with 
bits of desquamated epithelium and enormous numbers of cholera 
spirilla. The germs do not pass through the intestinal wall, but 
they multiply on and in the intestinal epithelium as well as in 
the intestinal contents. The general symptoms, shock, coma 
and the ultimate death, seem to be due in part to the absorption 
of poisons from the intestine and in part to the severe local irrita- 
tion in the abdomen. 


SPIRILLACEZ AND THE DISEASES CAUSED BY THEM 365 


The bacteriological diagnosis depends altogether upon the recog- 
nition of the cholera germ in the feces. During an epidemic of 
the disease a probable diagnosis in the individual case may be 
made by mere microscopic examination of stained preparations 
of the mucous flakes in the stools. The presence of abundant 
curved rods arranged parallel to each other is sufficient for a 
probable diagnosis. The problem presents itself in a different 
phase when it is necessary to recognize the first case of cholera in 
a given locality. Here it is necessary to follow up the microscopic 
diagnosis by cultures on gelatin plates, agar plates and in pepton 
solution, and the identification of the cultured organisms by ag- 
glutinating them with a known cholera-immune serum in high 
dilution (1 : 1000). The serum should be powerful enough in a 
dilution of 1 : 10,000 to agglutinate very definitely the culture 
used in producing it. The examination of immigrants for the detec- 
tion of cholera carriers also requires culture work. Thestool should 
be passed naturally, but a dose of salts is permissible if there is too 
great delay. About 1 gram of feces is mixed with so c.c. of sterile 
pepton solution! in a flask, and this is incubated at 37° C. for 
six to eight hours. A stained preparation is then made from the 
surface film of the flask. If no curved rods are found in it, the 
specimen is probably negative. A loopful of the surface film 
should nevertheless be transferred to a tube of pepton solution 
which is incubated for six hours and again examined microscopic- 
ally. If curved rods are found microscopically on the surface 
film of either the first or second culture, the problem of differentiat- 
ing between the cholera vibrio and other similar organisms is 
presented. Plate cultures on gelatin at 22° C. and on agar at 
37° C. should be made and at the same time the transplantation 
to fresh pepton solution should be continued at six-hour intervals. 
After eighteen hours, one examines the plates for typical colonies 
and subjects these to agglutination tests with specific serum of 
high titre. The bacteria from the surface film of the pepton solu- 
tion are also tested in the same way. A rapid clearing of the 

1 Pepton 10, NaCl 10, NaNO; 0.1, NaCO3 0.2, distilled water 1000. 


366 SPECIFIC MICRO-ORGANISMS 


microscopic field in the agglutination preparations warrants posi- 
tive diagnosis. 

Similar principles are followed in attempting to find cholera 
germs in drinking water. A solution of pepton 100 grams, salt 
roo grams, potassium nitrate 1 gram and sodium carbonate 2 
grams in distilled water tooo c.c. is prepared, filtered, distributed 
in ro flasks each of 1000 c.c. capacity, and sterilized. To each 
flask containing roo c.c. of this sterile solution, one adds about 
goo c.c. of the suspected water and incubates the mixture at 37° C. 
for six to eight hours. Subcultures and microscopic preparations 
are made from the surface films and any curved bacteria observed 
are tested as described above. 

The prophylaxis of cholera no longer rests upon the enforce- 
ment of quarantine regulations, for it is now known that conval- 
escents may carry the vibrio alive in their intestines for many 
weeks. The exclusion of the disease depends upon the bacterio- 
logical examination of every person coming from infected regions 
before he is allowed to land at his destination. A water-supply 
system well protected from fecal pollution is an element of safety _ 
for any community. The Hamburg epidemic of 1892 illustrated 
this point. The unfiltered water taken from the Elbe near the 
harbor carried the infection and distributed it throughout the city 
of Hamburg. In the presence of an epidemic the best protection 
against contact infection is provided by immunization. 

Ferran in 1884 first induced immunity to cholera in animals 
and in man by the subcutaneous injection of living cultures. 
Haffkine improved the method so as to make it reliable. He 
employed a first vaccine of attenuated virus and a second vaccine 
of high virulence with an interval of five days between the injec- 
tions. Kolle introduced the use of killed cultures, employing a 
single injection of 2 mg. of growth from an agar culture suspended 
in 1 c.c. of salt solution and killed by heating an hour at 58° C. 
As a result of this treatment the agglutinins, bacteriolysins and 


1 Krumwiede, Pratt and Grund, Journ. Infect. Diseases, 1912, Vol. X, pp- 
134-141. : 


SPIRILLACEAZ AND THE DISEASES CAUSED BY THEM 367 


opsonins for the cholera vibrio are increased. Practically such 
vaccination has resulted in a reduction in case incidence to about 
one-half and in mortality rate to about one-fourth that observed ~ 
among the unvaccinated. 

Spirillum (Vibrio) Metchnikovi.—This curved organism was 
found by Gamaleia in 1887 in the feces and in the blood of chickens 
suffering from enteritis. Morphologically and in cultures this 
organism resembles Sp. cholere very closely. It has a single 
flagellum. The growth and liquefaction of gelatin seems to be 
somewhat more rapid in the case of Sp. metchnikovt, and it usually 
produces a larger amount of indol. Accurate differentiation is 
possible only by animal experimentation and by testing with 
anti-sera. A minute quantity of culture of Sp. metchnikovi in- 
troduced into the skin of a dove or chicken is sufficient to cause 
general bacteremia and death, whereas even large doses (4 mg.) 
of true cholera organisms introduced into such a skin wound are 
without effect. Sp. metchnikovi is also much more virulent for 
guinea-pigs. Agglutination and bacteriolytic tests with specific 
sera also differentiate the two organisms. 

Spirillum (Vibrio) Finkler-Prior.—Finkler and Prior in 1885 
isolated this organism from the feces in cholera nostras. Morpho- 
logically it resembles the cholera vibrio very closely. Indol is 
not produced. It is apparently non-pathogenic. 

Spirillum Tyrogenum (Vibrio Deneke).—This organism was 
isolated from old cheese. It resembles the cholera vibrio but 
does not form indol and appears not to be pathogenic. 

A large number of other cholera-like organisms have been 
isolated in the various examinations for the cholera germ. Some 
of these can be differentiated morphologically, as they possess 
more than one flagellum. Others fail to produce indol or show 
other cultural difference from the true cholera organism. In 
some instances differentiation depends almost altogether upon 
the agglutination test. ‘This latter has come to be regarded as 
most important in the accurate recognition of the cholera organ- 
ism and its differentiation from other vibrios. 


CHAPTER XXV 
\ 
SPIROCHETE - 


Spirochzta Plicatilis.—Ehrenberg in 1833 observed this long 
slender spiral organism in swamp water. It occurs commonly 
in stagnant water among the alge which grow there and has also 
been found in sea water. The cell is about 0.75 in thickness and 
20 to 50ou in length. It moves by rotation and also by bending 
of the thread. Multiplication takes place by transverse division, 
sometimes occurring simultaneously at many points in a filament 
so that many short forms result. This organism is regarded as 
the type species of the genus Spirocheta. 

A number of saprophytic spirochetes are known. Dobell’ 
has made a careful study of several species, not only free-living ° 
but also parasitic spirochetes, directing special attention to their 
systematic relationships. He concludes that the spirochetes , 
belong to the bacteria and that they agree with the bacteria in 
their structure in all respects except the organs of locomotion. 
Concerning the flagella he seems to be doubtful. 

Spirocheta Recurrentis.—Obermeier in 1873 described the 
slender spiral organism first seen by him in 1868 in the blood in 
cases of relapsing fever. Ross and Milne observed a similar 
organism in man in Uganda in 1904 and Dutton and Todd in the 
same year demonstrated the presence of a spirochete in the blood 
in the African tick fever of the Congo. In 1905 a similar organ- 
ism was found in a case of relapsing fever in New York City. 
The disease has also been recognized in Russia, and in India. 
The spirochetes have been successfully inoculated into monkeys | 
and into rats, and various strains from different parts of the 
world have thus been made available for comparative study in 


1 Archiv. f.. Protistenkunde, 1912, Bd. XXVI, pp. “i 7-240. 
268 


wail 


SPIROCHATE 369 


the same laboratory. There are certain differences between 
these spirochetes of human relapsing fever, and several distinct 
varieties (or species?) are recognized. We shall consider them 
as varieties of Sp. recurrentis. 

Spirocheta Recurrentis var. Duttoni—This is the spirochete 
of Congo tick fever discovered by Dutton and Todd in 1904. It 
is about 0.454 in thickness and 24 to 30ou in length. The organism 
has been cultivated by Noguchi in ascitic fluid containing sterile 
tissue and covered by paraffin oil. The African tick fever caused 


Fic. 143.—Spirochezte of relapsing fever in blood of a man. (After Kolle and 
Wassermann.) 


by this organism is one of the most fatal of the relapsing fevers. . 


The tick remains infective for a very long time and also transmits 
the infection to its offspring through the egg. Other insects,? 
fleas‘and lice, are also capable of transmitting the infection. 

Spirocheta Recurrentis var. Rossii (Kochi).—This organism 
occurs in the blood of relapsing fever of East Africa. It resembles 
Sp. duttoni very closely. Noguchi obtained cultures readily in 
ascitic fluid containing sterile tissue. 

Spirocheta Recurrentis var. Novyi.*—This organism is 
more slender than the two preceding varieties, measuring about 


1 Journ. Exp. Med., 1912, Vol. XVI, pp. 199-210. 

2 Nuttall, Johns Hopkins Hosp. Bull., 1913, Vol. XXIV, pp. 33-39. 

3 Novy and Knapp: Journ. Inf. Diseases, 1906, Vol. III, pp. 291-393. 
24 i 


, 


370 SPECIFIC MICRO-ORGANISMS 


0.31 in thickness. The relapsing fever in which it occurs has 
been observed in South America. Noguchi has obtained cultures 
by the same methods as he employed for Sp. rossii, but the cultiva- 
tion is more difficult. 

Several other varieties df spirochetes, which cause ay 
fever in man, have been recognized. The spirochete concerned 
in any case seems to be able to infect several species of insects and 


FIG. 144.—Spirocheta recurrentis (novyi). Organisms of different lengths in the 
blood of a white rat. X1500. (After Novy and Knapp.) : 

to be transmitted to a new mammalian host by them. Further: 

more one species of insect seems to be capable of transmitting 

‘any one of these spirochetes.! 

The diagnosis of relapsing fever depends upon recognizing the 
characteristic spirochetes in the blood during the febrile attack. 
Their recognition offers little difficulty, as a rule, but they may be’ 
overlooked by a beginner. In doubtful cases it is well to search 
the fresh drop of blood not only by direct central illumination 


1 Nuttall: Johns Hopkins Bull., 1913, Vol. XXIV, pp. 33-39. 


SPIROCHETE 371 


with a yellow light but also by means of dark-field illumination , 
and to examine thin films made by mixing India ink 3 parts with 
the blood 1 part and spreading very thin. Finally thin blood films 
should be stained and examined. Theinoculation of white rats with 
1 to 5 c.c. of blood conveys the infection to them and the parasites 
appear in the blood of the animal 2 to 4 days after inoculation. 
The spirochetes may vanish from the blood with marvelous rapidity. 

Spirocheta Anserina.—Sacharoff in 1890 discovered this 
spiral organism in the ‘blood of geese suffering from a serious 
disease in the Caucasus. Ducks and chickens are also susceptible. 
The spirochete is about 0.5 thick by 10 to, 204 long. It is con- 
sidered by Nuttall to be identical with the Sp. gallinarum of 
Marchoux and Salimbeni. 

Spirocheta Gallinarum.—Marchoux and Salimbeni’ in 1903 
discovered this organism in the blood of diseased chickens at 
Rio Janeiro. The organism is 0.54 thick and 15 to 20 long. 
The disease is transmitted by means of the fowl tick Argas minia- 
tus (persicus?), most effectively when the tick is kept at a tempera- 
ture of 30° to 35° C. In cold climates the disease is unknown. 
Leishman and Hindle have studied very carefully the changes 
which the spirochetes pass through in the body of the insect. 
They found numerous exceedingly minute “coccoid bodies” in 
the cells of the Malpighian tubules. These minute bodies are 
considered! to be the products of a fragmentation of spirochetes 
and to be capable of again growing into typical spirochetes. If 
the: view is correct these bodies necessarily play an important part 
in the infection of the vertebrate host and in the inheritance of 
the infection in the insect species. 

Spirocheta Muris.—This is a very short spirochete which 
occurs naturally in a non-fatal relapsing fever of rats and mice. 
It possesses one or sometimes two flagella on each end and multi- 
plies by simple transverse fission. The infection in rats and prob- 
ably also in mice is evidently world wide. 


' Nuttall: Harvey lecture, 1913. 


372 SPECIFIC MICRO-ORGANISMS 


. In 1915, Futaki and Takaki! found a spirochete apparently 
identical with Spirocheta muris in rat-bite fever (Sodoku) of man, 
To the organism they gave the name Spirocheta morsus-muris, 


Fic. 145.—Spirocheta (morsus) muris in lung of mouse inoculated with blood 
from human rat-bite fever. Silverimpregnation. X1500. (After Futaki, Takaki, 
Taniguchi and Osumi.) : 


The disease assumed some importance in the armies during the 
period of trench warfare. It is transmissible to various animals, | 


Fic. 146.—Spirochata (morsus) muris in blood of guinea-pig with experimental. . 
rat-bite fever. Giemsa’s stain. 1250. (Afler Futaki and associates.) : 


e 


the guinea-pig being perhaps most satisfactory for study of the 
experimental disease. 

1 Futaki, Takaki, Taniguchi and Osumi: Journ. Exp. Med., 1916, 23, p. 249; 
ibid., 1917, 25, P- 33- ‘ 


SPIROCHATE 373 


Spirochzta (Leptospira) Icteroheemorrhagie.— This organism 
was discovered by Inada and Ido! in the human disease known as 
infectious jaundice or Weil’s disease. The organism is a long 
slender somewhat irregular spiral. It occurs in the blood and in 
greater abundance in the substance of the kidney. It grows as 


\ 


Fic. Fan melebisebindg icterohemorrhagi@ in section of human liver. Drawing of 
a silver-impregnated specimen. (After Inada, Ido, Hoki, Kaneko and Ito.) 
an aérobe in diluted rabbit serum at temperatures ranging from 
10° to 37° C., which suggests that insects may act as reservoirs 
for the virus.?_ Wild rats frequently harbor the parasite within 
their kidneys and excrete it with the urine. The mode of transfer 


‘Inda, Ido, Hoki, Kaneko and Ito: Journ. Exp. Med. 1916, 23, p. 377. 
2 Noguchi: Journ. Exp. Med., 1918, 27, pp. 575, 593, 609. 


376 SPECIFIC MICRO-ORGANISMS 


blood (black vomit). It is frequently fatal. Permanent im- 
munity follows recovery. Reed, Carroll, Lazear and Agramonte,' 
in Igor, showed that the virus is present in the blood at least 
during the first two or three days of the attack, that it will pass 
through a porcelain (Chamberland B) filter and that it is naturally 
transmitted from man to man by the mosquito Aédes (Stegomyia) 
calopus, which becomes capable of inoculating the disease about 
twelve days after sucking blood which contains the virus. The 
mosquito probably remains infective as long as it lives and may be 
regarded as an essential agent in the spread of yellow fever. Pro- 
phylacticmeasures based upon this deduction have been remarkably 
successful in the suppression of the disease. . 

The newer work of Noguchi suggests that there may be verte- 
brate animals which serve as reservoirs of the yellow fever virus 
in the tropics. 

Spirochzta (Leptospira) Hebdomadalis.—Ido, Ito and Wani? 
have found this organism, which resembles Spirocheta ictero- 
hemorrhagie in form and motion, in blood of patients suffering 
from the Japanese seven-day fever, Nanukayami. It has been 
successfully inoculated into young guinea-pigs. 

Spirochzta Gallica.—Couvy and Dujarric de la Riviére* have 
found a small spirochete in the blood in trench fever and have 
suggested the name Spirocheta gallica for it. They also found the 
spirochete in the liver and kidneys of guinea-pigs inoculated with 
the human blood. Their results suggest the probable causal 
relationship.of this organism to trench fever, but critical confirma- 
tion has not yet appeared. 

Trench fever [Febris quintana (Wolhynica)] was probably 
the most important epidemic disease of the world war up to 1918. 
MacNee in 1915 proved that it was transmissible by blood in- 


1 The publications of Reed, Carroll and their associates have been issued as a 
volume entitled Yellow Fever, U. S. Senate Document No. 822, 61st Congress 
3rd Session, 1911. 

2Tdo, Ito and Wani: Journ. Exp. Med., 1919, 29, p. 190. 

3 Comptes rendus Soc. Biol., 1918, 81, p. 22. 


SPIROCHATA 377 


oculation. Early in 1918 | the Trench Fever Commission,’ 
Medical Research Committee, American Red Cross, confirmed the 
inoculation experiments of MacNee and demonstrated that the 
virus resides in greatest concentration in the blood plasma and that 
it is transmitted from man to man by natural infestation with 
body lice (Pediculus humanus). The direct inoculation experiments 
described in detail by Baetjer? and the louse transmission ex- 
periments described in detail by MacNeal and Peacock® leave 
no room for doubt upon these points. This portion of the work of 
the commission has been confirmed in all essentials by the simul- 
taneous and independent research of the Research Committee* 
of the British War Office, working in England under the presidency 
of Sir David Bruce. 

Spirochzta Pallida (Treponema Pallidum).—Schaudinn and 
Hoffmann in 1905 observed this slender spiral organism in pri- 
mary syphilitic lesions, in fluid obtained from swollen lymph glands 
in syphilis and in the liver and spleen of a still-born syphilitic 
fetus. The occurrence of the organism in syphilitic lesions was 
quickly and abundantly confirmed by other workers. Cultures 
were obtained in collodion sacs by Levaditi and McIntosh in 1907. 
Schereschewsky, and Muhlens and Hoffman obtained cultures 
in gelatinized horse serum. Noguchi’ has carried out the most 
successful cultural work and has succeeded for the first time in 
causing syphilitic lesions in animals by the inoculation of pure 
cultures. 

Sp. pallida occurs naturally only in human syphilis. It is a 
slender spiral 0.2 to 0.35u in thickness and 3.5 to 15.5 in length. 
Its curves are narrow and very regular. It is actively motile, 


t 


1 Strong, Swift, Opie, MacNeal, Baetjer, Pappenheimer and Peacock: Medical 
Bulletin, Am. Red Cross, March, 1918, 1, p. 376; Trench Fever, Oxford University 
Press, 1918, pp. 446 +VIII. 

2 Trench Fever, Oxford Press, 1918, Chapter VII, p. 61-74. 

3 Trench Fever, Oxford Press, 1918, Chapters X and XI, p: 143-274. 

4Transmission of Trench Fever by the Louse: British Med. Journ., Mar. 23, 
1918. : 

5 Journ. Exp. Med., 1911, Vol. XIV, p. 99; 1912, Vol. XV, p. go. 


3 78 SPECIFIC MICRO-ORGANISMS 


as are all the spirochetes, and has a very slender flagellum at each 
end. The usual motion is that of rapid rotation on the longitudinal 
axis with progression, but at times there is gross bending of the 
filament, especially when the organism is living under unfavorable 
conditions. The mode of division 
is a somewhat vexed question as it 
is in regard to the whole group of 
spirochetes. Transverse and longi- 
tudinal division have been de- 
scribed. Probably the weight of 
authority! now favors transverse 
division as the sole mode of mullti- 
plication, although able adherents 
' to the opposite view are not lack- 
ing. The refractive index of the 
filament is not very much greater 
than that of serum, so that the 
unstained organism is difficult ta 
see by direct illumination. Dark- 
field illumination is more satis- 
factory. Sp. pallida in film prep- 
arations stains with difficulty by 
Fic. 151.—Film preparation from Ordinary methods. Schaudinn em- 
a genital syphilitic Papule; in the ployed Giemsa’s modification of 
center are two specimens of Spiro- . 
cheta pallida, the other three are the Romanowsky stain. Good re- 
szcaimens of, Sptrochets reringens. sults are obtained by staining 
with solutions of the Romanowsky 
staining principles in methyl alcohol provided an excess of me- 
thylene-violet be present (see p. 44). Tunnincliff? recom- 
mends staining with a mixture of saturated alcoholic solution of 
gentian violet, 1 part, in 5 per cent carbolic acid, 9 parts. Thin 
films are essential but staining process requires only a few seconds. 


1 Journ. Exp. Med., 1911, Vol. XIV, p. 99; 1912, Vol. XV, p. go. 
2 Journ. A. M. A., 1912, Vol. LVIII, p. 1682. 


. SPIROCHATE 379 
Probably the most satisfactory stain is that of Fontana.' In pieces 
of tissue the spirochete is best stained by the method of Levaditi. 
For this purpose thin (1 mm.) pieces of syphilitic tissue are fixed 
in formalin (10 per cent) for 24 hours or longer and hardened in 
95 per cent alcohol for a day. The alcohol is then removed by 
soaking in distilled water and the tissue is transferred to a fresh 
1 to 3 per cent solution of silver nitrate in distilled water. This 
is placed at 37° C. in the dark for three to five days. The tissue 
is next washed in distilled water and placed in reducing fluid, 
consisting of pyrogallic acid 3 grams, formalin (40 per cent for- 
maldehyde) 5 c.c. and distilled water 100 c.c., for one to two days. 
It is then washed in distilled water, dehydrated, embedded in 
paraffin and sectioned. The spirochetes are stained a dense 
black by this method. The sections may be stained to show 
histological structure also, by applying methylene blue or toluidin 
blue to them after they have been fixed on the slide. 

Cultivation of Sp. pallida has been most successfully practised 
by Noguchi.? He has grown the organism in a mixture of serum 
and water, to which naturally sterile tissue was added, and in 
ascitic-fluid agar with similar bits of tissue, always under strict 
anaérobic conditions. The technic of culture is somewhat diffi- 
cult and the original papers should be consulted in detail. Inocu- 
lation of the cultures into rabbits and monkeys has caused typical 

‘ syphilitic lesions. 

Noguchi’s luetin is prepared by grinding the solid medium 
rich in spirochetes in a mortar and emulsifying it in a small 
amount of fluid. This is then heated to 60° C. for an hour and 
preserved by the addition of 0.5 per cent carbolic acid. The 
final preparation contains many dead unbroken spirochetes. 


1 Three solutions are required (a) Glacial acetic acid 1, formalin 20, distilled 
water 100; (b) Phenol 1, tannic acid 5, water 100; (c) Silver nitrate (25 per cent 
solution) 5 c.c., ammonia water 1 drop. The thin smear is dried inair. Treat with 
(a) one minute; wash in water; cover with (b) and steam one-half minute; wash 
in distilled water; cover with (c) and steam one-half minute; wash; biot; dry; mount 
in balsam. Medical War Manual No. 6, U. S. Army, 1919, p. 30. 

2 Journ. Exp. Med., 1911, Vol. XIV, p. 99; 1912, Vol. XV, p. go. 


380 SPECIFIC MICRO-ORGANISMS 


Syphilis is an inoculation -disease which has been widely 
prevalent throughout the civilized world since the early part of 
the 16thcentury. Transmission takes place by direct contact and 
in the great majority of instances by venereal contact, although 


Fic. 152.—Spirocheta pallida stained by Levaditi method. The section shows 
an infarcted lymph vessel at the junction of two branches. The lumen is filled with 
leukocytes. The spirochetes follow the lymph vessel for the most part, but are also 
penetrating into the surrounding tissue. (From Doflein after Ehrmann.) 
many authentic cases of transmission by means of intermediate 
objects are known. The spirochete is able to live for some hours 
outside the body if drying is prevented. The primary lesion 
develops at the point of inoculation about two weeks after that 
event, first as a papule, which becomes vesicular and. ulcerates, 


SPIROCHATA. 381 


remaining indolent for several weeks. The neighboring lymph 
glands become swollen. The secondary manifestations occur 
about a month later as a general macular or sometimes papular 
eruption on the skin, together with sore throat and ulcerated 
patches in mouth. The skin eruption does not itch. Subsequent 
to this stage there may be local necrotic lesions (gummata) in 
various parts of the body, or low-grade inflammatory changes in 
the meninges and central nervous system. Bacteriological 
methods of diagnosis are of assistance in some cases in all the 
various stages of syphilis. Early in the disease the spirochetes 
are relatively numerous, in certain locations at any rate, while 
later the parasites may be so few as to render their detection 
practically hopeless for diagnostic purposes. In these later 
stages, however, the presence of specific and other antibodies in 
the body fluids of the patient may often be recognized and this 
recognition employed as an aid in diagnosis. 

Microscopic examination of a primary ulcer is best done by 
means of the dark-field illumination. For this purpose the ulcer 
(which should not have been treated with mercurials) is carefully 
cleansed and a few drops of freshly exuded serum collected in a 
glass capillary, and the usual slide-cover-glass preparation is made 
with this fluid. Permanent preparations are made most easily 
by mixing such serum with India ink on a slide and spreading 
the mixture in a very thin layer. Collargol, one part in nineteen 
parts of water, gives even more satisfactory preparations! than 
India ink. It is used in the same way. Thin films of the serum 
on slides or cover-glasses may be stained as directed above. Micro- 
scopic examination of fluid obtained by gland puncture or from 
secondary lesions on the skin or mucous membranes is carried 
out in the same way. Serious confusion in the recognition of the 
spirochete is likely to arise in the case of lesions in the mouth or 
pharynx, inasmuch as some of the normal mouth spirochetes are 
very similar in form to Sp. pallida. The presence of typical 
spirochetes in the juice aspirated from a lymph gland is practically 

1 Harrison : Journ. Roy. Army Med. Corps, 1912, Vol. XIX, p. 749. 


382 SPECIFIC MICRO-ORGANISMS 


diagnostic, and the recognition of typical organisms in genital 
chancres or lesions on the skin has important diagnostic value. 

Inoculation of animals is of little practical use in diagnosis, 
but it has been possible by this method to demonstrate the fre- 
quent presence of Sp. pallida in the circulating blood in cases of 
untreated secondary syphilis. 

The detection of antibodies in the blood of the patient is under- 
taken in two ways, first by the complement-fixation (Wassermann) 
test and second by the luetin test. For the complement-fixation! 
test, as performed at the Laboratories of the New York Post- 
Graduate Medical School and Hospital the following are employed: 

t. The red blood cells are obtained by defibrinating fresh 
sheep’s blood, filtering it through paper if necessary to remove 
fragments of clot, separating the cells in the centrifuge and wash- 
ing them four times with o.9 per cent salt solution. Finally 1 c.c. 
of the corpuscles as packed by the centrifuge is suspended in 19 
c.c. of o.g per cent salt solution; 0.2 c.c. of this suspension is 
arbitrarily taken as the unit of red blood cells. 

2. The complement is obtained by drawing 5 to 10 c.c. of 
blood from a large guinea-pig by cardiac puncture. This blood 
is transferred to a Petri dish, allowed to clot, incubated at 37° C. 
for 30 minutes and then refrigerated. The separated serum is 
then drawn off with a pipette and 2 c.c. of it are mixed with 18 c.c. 
of cold 0.9 per cent salt solution. This 10 per cent solution of 
guinea-pig’s serum is kept insa cold place, preferably immersed in 
ice water. It is prepared on the day it is to be used. The unit 
of complement is contained in 0.1 c.c. of this solution. Two 
units (0.2 c.c.) are employed in the actual test. 

3. The hemolytic amboceptor is prepared by injecting 2 c.c. 
of thoroughly washed (five times) sheep’s corpuscles intravenously 
into a large rabbit at intervals of three days, until four injections 
have been given. Ten days after the last injection the animal is 
allowed to fast for 12 hours and the blood is then aseptically 


* Smith, J. W., and MacNeal: Journ. Immunology, 1916, 2, p. 75; Journ. Infect. 
Diseases, 1917, 21, 233. 


SPIROCHATE 383 


drawn from the carotid artery, allowed to clot and the serum 
separated by standing at 37° C. for two to five hours. The clear 
serum is transferred to small glass ampoules in amounts of 0.5 to 
1.0 c.c. and hermetically sealed. These are then heated at 56° C. 
for 30 minutes and stored in the refrigerator. The hemolytic 
power of this serum is ascertained by titration. The unit is that 
amount which, when mixed with 0.2 c.c. (1 unit) of corpuscles 
and o.1 c.c. (r unit) of complement and sufficient salt solution 
_ (0.9 per cent) to make a total volume of 1 c.c., will cause complete 
laking of the red blood cells in exactly 1 hour after being placed 
in the incubator (water bath) at 37° C. The unit of amboceptor is 
ordinarily contained in o.1 c.c. of a-dilution of 1 part of serum in 
500 to 1200 parts of salt solution. After the strength has been 
ascertained by trial of a series of different quantities of a strong 
(1:200) solution, the amboceptor dilution to be used on the same 
day is made up so that o.1 c.c. contains x unit. 

‘The amboceptor is quite permanent under ordinary refrigera- 
tor conditions, but when diluted it may deteriorate after a few 
days. The relation of complement, red blood cells and ambo- 
ceptor is tested always immediately before undertaking a comple- 
ment-fixation test. If the mixture of one unit of each of these in 
a total volume of 1 c.c. produces’ complete hemolysis at the end 
of an hour, the hemolytic system is considered satisfactory. If 
there is only a slight discrepancy this may be corrected by altering 
the final dilution of the amboceptor from its usual strength, within 
limits of 25 per cent. If the discrepancy is greater than this it is 
well to obtain a new sample of complement or of sheep’s cells or 
of both. The hemolytic system should behave much the same 
from day to day when the technic is accurate. 

4. The patient’s serum is obtained from 5 to 10 c.c. of blood 
drawn from the elbow vein. The serum should be removed from 
the clot within 24 hours, freed from corpuscles and preserved with 
chloroform unless it can be tested promptly. Particular care is 
required when the serum is to be sent by mail. The serum is 
heated at 54° to 56° C. for 30 minutes just before use. 


384 SPECIFIC MICRO-ORGANISMS 


s. The antigen is an alcoholic extract of. the heart muscle of 
beef. The clean, finely chopped heart muscle, ro grams, is ex- 
tracted in 100 c.c. of absolute ethyl alcohol at 37° C., with fre- 
quent shaking for two weeks. It is filtered through paper. The 
filtrate is then refrigerated a day and again filtered. This clear 
filtrate is the plain alcoholic antigen and it is sealed up in small 
ampoules or stored in a tightly stoppered bottle. The strength of 
antigen to be used must be ascertained by careful titration. 
A dilution of 1 c.c. of the antigen in 9 c.c. of salt solution is first 
prepared. Then various quantities, 0.1 C.c., 0.2 C.C., 0.3 C.C., 
0.4 c.c. and o.5 c.c. of this suspension are placed in separate tubes. 
To each tube is added 2 units of complement and sufficient salt 
solution to bring the total volume to 0.6c.c. The tubes are placed 
in the ice-box overnight. Then one unit of corpuscles (0.2 c.c.) 
and two units of hemolytic amboceptor (0.2 c.c.) are added and the 
tubes are incubated an hour at 37° C. Of those tubes in which 
hemolysis is not complete, the one containing the least antigen 
marks the concentration at which the antigen is distinctly anti- 
complementary. The second test of the antigen is now under- 
taken. Various amounts of ar to 100 dilution, 0.01 C.c., 0.03 .c., 
0.05 C.C., 0.1 c.c and 0.2 c.c., are measured into tubes. To each 
tube is then added 2 units of complement, 0.02 c.c. of serum from 
an active untreated case of syphilis and sufficient salt solution to 
make a total volume of 0.6 c.c. The tubes are left overnight in 
the ice-box. Then 1 unit of corpuscles (0.2 c.c.) and 2 units of 
hemolytic amboceptor (0.2 c.c.) are added and the tubes are 
incubated one hour at 37° C. Of the tubes showing no hemolysis 
(complete fixation), that one which contains the least antigen 
marks the lowest effective concentration of the antigen. This 
amount of antigen should be very much less than the anti-com- 
plementary amount ascertained in the first test. Ordinarily 
it is about 14009 of this amount. The unit of antigen to be em- 
ployed should be chosen so that it is several times greater than 
the least effective quantity but still not more than one-fifth to 
one-fourth the least anti-complemientary amount. Having chosen 


SPIROCHATA 385 


the tentative antigen unit, a third test is applied. One, two and 
four units of antigen are placed in tubes and a unit of corpuscles 
is added to each, together with sufficient salt solution to make the 
total volume 1 c.c., and these are incubated for an hour. The 
corpuscles should not be laked. If they are laked the antigen is 
itself markedly hemolytic. A satisfactory antigen should per- 
form its specific function of fixing complement in the presence 
of a syphilitic serum in an amount which is at most 1é9 of the 
amount which is in itself either anti-complementary or hemolytic. 
It keeps well in: the refrigerator as the alcoholic solution. The 
dilution for use should be freshly prepared by slowly adding the 
salt solution, a few drops at a time, to the alcoholic antigen in a 
test tube, with thorough shaking. 

The antigen is the element in the test which is designed to 
enter into chemical reaction with the specific substance in the 
patient’s blood, which is present there as a result of active syphilis. 
During the course of this reaction, complement is absorbed 
or destroyed. The nature of the lipoidophilic substance? is un- 
known. It behaves in the test very much as a specific immune 
body would be expected to behave. Experience has shown that an 
antibody of this nature is rarely present in other conditions 
than active syphilis and that it is present in this disease. Upon 
the results of this experience we have to rely in ascribing diagnos- 
tic value to the test. 

In performing a test for diagnosis, sera from several patients 
should be tested at the same time, and one, two or three sera, pre- 
viously tested and found to fix complement in varying degrees, 
and at least one serum known to give a negative result, should 
be tested along with the new samples. Three tubes are used for 
each serum to be tested. The first incubation is carried out in the 
refrigerator over night and the second incubation, after the addi- 
tion of the sensitized cells is at 37° C. in the water bath. 


1The anticomplementary test upon one, two, three and four units of antigen 
must be repeated every time a diagnostic test is‘carred out and simultaneously 
with it using the identical reagents. ‘ 
- *Simon: Infection and Immunity, Phila., 1912, p. 272. 
25 


386 SPECIFIC MICRO-ORGANISMS 


Tube No. 1 Tube No. 2 ' Tube No. 3 
Complement 2 units Complement 2 units Complement 1 unit 
(0.2 c.c.) (0.2 C.c.) (0.2 ¢.c.) 
Patient’s serum o.I C.c. Patient’s serum 0.02 c.c._ || Patient’s serum 0.05 c.c. 
Salt solution 0.3 c.c. Antigen 1 unit (0.1 c.c.) Antigen 1 unit (0.1 c.c.) 
Salt solution 0.28 c.c. Salt solution 0.25 c.c, 


Mix thoroughly and leave overnight in ice box. Then add: 


Sheep’s corpus- } Sheep’s corpus- Sheep’s corpus- 

cles 1 unit cles 1 unit cles 1 unit 

(0.2 C.c.) 0.4€.C.1] (0.2 €.c.) 0.4.€.C.4]| (0.2 c.c.) 9.4.¢.€.) 
Hemolytic am- Hemolytic ambo- Hemolytic ambo- 

boceptor 2 ceptor 2 units ceptor 2 units 

units (0.2 c.c.) (0.2 c.c.) (0.2 €.Cc.) 


Mix thoroughly and incubate at 37° C. in water bath for 1 hour, recording the 
progress of hemolysis at intervals of 15 minutes. Then refrigerate 16 hours and 
record the final reading. 


Tube No. 1 should show complete hemolysis early in the second 
incubation. If this has behaved properly and the tests on the 
known sera have resulted as they did when previously tested, 
then the behavior of Tubes 2 and 3 is a measure of the amount of 
lipoidophilic suhstance in the serum of the patient. One dis- 
tinguishes six different grades of reaction, from complete fixation 
(no trace of hemolysis) to no fixation (complete hemolysis). 
These are designated by the signs (+ +++) (+++) (++) 
(+) (4) and (—). 

The luetin test is performed by injecting 0.05 c.c. of luetin 
intracutaneously in two places on the left arm and at the same 
time 0.05 c.c. of a control suspension, consisting of the medium 


‘ The suspension of sheep’s corpuscles containing 1 unit in o.2 c.c. and the solu- 
tion of hemolytic amboceptor containing 2 units in 0.2 c.c. are quickly mixed to- 
gether in equal parts, and 0.4 c.c. of this homogeneous mixture is added at this point. 
This procedure results in a saving of time as well as greater accuracy. 


SPIROCHETE , 387 


without any growth of spirochetes, at two points on the right 
arm. Local inflammation on the left arm, appearing in two to 
ten days and sometimes resulting in the formation of a pustule, is 
regarded as a positive test. The test is often negative in the 
earlier stages of syphilis. 

The various diagnostic tests for syphilis are now extensively 

‘employed. Microscopic search for the spirochete is of value in the 

untreated primary and secondary stages. The complement-fixa- 
tion test becomes positive a few weeks after the appearance of the 
primary lesion and is generally regarded as indicating an active 
syphilitic process. The luetin test may be positive in latent or 
inactive ‘syphilis when the Wassermann is negative. Further 
experience with the luetin test is necessary in order to determine 
its real significance. .- 

Spirocheta (Treponema) Refringens.—This is a relatively 
gross spirochete which occurs in primary syphilitic lesions along 
with Sp. pallida. It seems to have no pathogenic properties. 
Noguchi! has obtained pure cultures of it and found them with- 
out pathogenic properties for rabbits and monkeys. 

Spirocheta (Treponema) Microdentium.’—This is one of the 
common spirals of the mouth. It may be confused with Sp. pal- 
lida, which it resembles in size and shape. Pure cultures have 
been obtained by Noguchi. Other spirochetes of the mouth 
have also been cultivated by this investigator and there are prob- 
ably several species of them. 


1 Journ. Exp. Med., 1902., Vol. XV, p. 466. 
2 Noguchi: Journ. Exp. Med., Vol. XV, pp. 81-80. 


CHAPTER XXVI 
THE FILTERABLE MICROBES 


The Virus of Foot-and-mouth Disease.—This filterable or- 
ganism occurs in the vesicles present in the mouth and on the 
feet of the diseased animals, and also in the milk of cows suffering 
from foot-and-mouth disease. The virus was shown to be filter- 
able by Loffler and Frosch in 1898. It is rendered inert by heat- 
ing to 50° C. for ro minutes. Animals are immune after recovery 
from the disease. Cattle and swine are naturally susceptible 
and a few cases of the disease have occurred in man. Nothing 
definite is known concerning morphology or cultures. The in- 
fection seems to be transmitted with the food as well as by 
inoculation. . 

The Virus of Bovine Pleuro-pneumonia.—This organism is 
present in the affected lungs and in discharges from the respira- 
tory tract of cattle suffering from pleuro-pneumonia. Nocard 
filtered the virus through a Chamberland ‘“‘F” filter in 1899. It 
is rendered inert by heating at 58° C., but retains its virulence in 
glycerine for weeks and resists freezing. Cultures have been 
obtained by the collodion-sac method byNocard and Roux. The 
organisms in such cultures are extremely minute and variable in 
form. Some of them are spirals and others approximately spher- 
ical. Immunity follows recovery from the disease, and has been 
induced artificially by inoculation with cultures and also by inocu- 
lation with virulent exudate from the lung of a dead animal into 
the subcutaneous tissue of the tail of the animal to be immunized. 

The Virus of Cattle Plague (Rinderpest).—This organism 
occurs in the blood, organs and excretions of cattle suffering from 
the disease. It was shown to be filterable by Nicolle and Adil- 


1 Kolle and Wasserman, Handbuch, 1012. Bd. I. S. 028. 


THE FILTERABLE MICROBES 389 
} 


Bey in 1902, and is able to pass through the Chamberland ‘“‘F”’ 
filter. The virus resists drying for four days and remains active 
for two or three months when spread on hay in a dark place. 
It is destroyed by distilled water in five days, by glycerin in eight 
days and rendered avirulent in a few hours by admixture of bile. 
The disease is an acute febrile disorder characterized by severe 
inflammation of the mucous membranes and rapid emaciation. 
It is usually fatal. Immunity follows recovery and is induced 
artificially by injecting the bile of infected animals under the 
skin of the healthy cattle. In this way an active immunity is 
acquired without an evident attack of the disease. 

The Virus of Rabies.—This organism exists in the central 
nervous system, the peripheral nerves, the salivary glands, the 
saliva and less frequently in other parts of the body of persons 
or animals suffering from lyssa or rabies. The virus was filtered 
by Remlinger in 1903. It may also be dialyzed through collodion 
sacs.! The virus is rendered inert by drying for two weeks, and 
by heating at 55° C. for 30 minutes, by admixture of bile in a few 
minutes, and by the gastric juice in 5 hours. It remains virulent 
in glycerine for several months. Negri in 1903 described certain 
bodies which seem to occur in the central nervous system in- 
variably and exclusively in this disease. They are especially 
numerous in the ammon’s horn of the brain in cases of street 
rabies. Preparations should be made from the gray matter of 
the brain. A bit of this tissue is carefully spread on a slide by 
exerting moderate pressure upon it with a second slide or a cover- 
glass and at the same time moving it along the surface of the first 
slide. The film is fixed in pure methylic alcohol and stained with 
Giemsa’s solution, or it may be stained directly without fixation 
with Leishman’s stain. The Negri bodies are round and some- 
what irregular in outline, from rp to 27u in diameter, and usually 
inside the nerve cells. In the interior of the larger bodies, smaller 
spherical structures of variable size and number may be seen. 
The exact nature of the Negri bodies is uncertain. Some stu- 

1 Poor and Steinhardt, Journ, Infect. Dis., 1913, Vol. XII, pp. 202-205. 


390 SPECIFIC MICRO-ORGANISMS 


dents of rabies regard them as protozoa, while others consider 
" them to be products of cell degeneration. The evidence to de- 
cide the matter is not yet at hand. They seem to occur only in 
rabies and to be constantly present in this disease. 


Fic. 153.—Section through the cornu ammonis of brain of a rabid dog; stained by 
the method of Lentz. Five Negri bodies of different sizes are shown, enclosed within 
the ganglion cells. The smallest contains only three minute granules. (After Lentz,’ 
Centralbl. f. Bakt, 1907, Abt. I, Vol. XLIV, p. 378.) 


Lyssa or rabies! is primarily a disease of dogs but it occurs in 
other mammals as well, usually as a result of dog bites. In ani- 
mals inoculated directly into the brain with the most virulent 
material (fixed virus), the symptoms of rabies appear in 4 to 6 
days and death occurs on the seventh day. Inoculation with the 
saliva or nervous tissue of a mad dog (street virus) rarely causes 


1 For a general discussion of rabies see Cumming: Journ. A. M. A., 1912, Vol. * 
LVIII, pp. 1496-1499. 


THE FILTERABLE MICROBES 3901 


symptoms before three weeks and the onset may be delayed for 
a year. In fact many persons and animals bitten by rabid dogs 
may fail to develop the disease at all. This variability depends 
upon the virulence and the amount of virus and especially upon 
the part of the body into which it is introduced. Bites upon the 
face or hands, because of the rich nerve supply and the lack of 
protection by clothing, are especially dangerous. After the dis- 
ease has developed so as to cause symptoms, death is inevitable 
in the present state of our knowledge. 

Rabies may be diagnosed in an animal by observing the course 
of the disease, by autopsy and by inoculation of test animals and 
observation of the course of the disease in them. If the sus- 
pected animal be caged, the question of rabies may be settled in a 
few days, for, if he is mad, the raging stage will be quickly followed 
by the characteristic paralysis and death. If the animal has been 
killed, a careful autopsy may reveal the absence of food from the 
digestive tract and the presence there of abnormal ingested ma- 
terial (grass, wood or stone), highly suggestive of rabies. Mi- 
croscopic examination of the central nervous system may reveal 
the Negri bodies, characteristic of the disease. For confirmation 
of the diagnosis a portion of the brain or spinal cord, removed with- 
out contamination, should be injected into the brain of guinea- 
pigs and rabbits and the effects observed. This last test 
carried out by an experienced observer is the most trustworthy 
of all. 

The Pasteur treatment of rabies is designed to induce immu- 
nity after the person has been bitten and before the disease has had 
time to develop. Pasteur! first demonstrated the possibility of 
this by experimental work on dogs, and the subsequent use of 
the method in man has been remarkably successful and the dis- 
ease is practically always prevented if the treatment is begun 
directly after infliction of the infecting wound. The first essen- 
tial is thorough cauterization of the wound, best with concentrated 
nitric acid under anesthesia. The patient is then injected sub- 

‘Vallery-Radot: The Life of Pasteur, 1911, Vol. II, p. 188. 


392 SPECIFIC MICRO-ORGANISMS 


cutaneously with emulsions of the spinal cords which have been 
removed from rabbits dying of rabies after inoculation with the 
fixed virus, and which have been dried by hanging in bottles 
over caustic soda for some time. The first injection is prepared 
from cords hung for 14 and 13 days, the second from cords hung 
12 and 11 days, and so on until the three-day cord is reached on 
the seventh or eighth day of the treatment. The series from 
five-day down to three-day cords is then repeated several times, 
the whole treatment lasting about 21 days. The course of treat- 
ment is varied somewhat according to the urgency of the case and 
the severity of the wounds inflicted. It is most effectively carried 
out at special Pasteur institutes devoted to this work, but the 
material for injection may be shipped for some distance when 
necessary. 

The Virus of Hog Cholera.—Dorset, Bolton and McBryde, 
continuing the investigations of de Schweinitz, demonstrated in 
1905 the presence of a filterable agent in the blood of hogs suffering 
from hog cholera, capable of causing the disease upon injection 
into healthy animals. It passes through the Chamberland ‘B” 
and “F” filters. It leaves the body in the urine and probably 
also in other excretions, and seems to enter the new victim with 
the food and drink. The virus resists drying for three days, 
remains alive in water for many weeks and in glycerine for eight 
days. It is destroyed at 60° to 70° C. in an hour. 

King, Baeslack and Hoffman! have found a short, rather thick, 
actively motile spirochete, Spirocheta suis, in the blood in forty 
cases of hog cholera, together with abundant granules which may, 
perhaps, represent a stage of this organism. The spirochete has 
not been found in healthy hogs. It seems probable that this 
organism may prove to be the causative agent of the disease, but 
further evidence is necessary to demonstrate this relationship. 

Hog cholera is an extremely contagious disease of hogs, fre- » 
quently fatal, characterized by fever and by ulcerations in the 
intestine. Immunity follows recovery and is induced artificially 

1 Journ. Infect. Dis., 1913, Vol. XII, pp. 30-47; Dp. 206-225. 


THE FILTERABLE MICROBES 393 


by the injection of serum from a hyperimmune hog (passive 
immunity) and by the injection of such serum together with viru- 
lent blood from a hog sick with the diseaSe (combined passive 
and active immunity). 

The Virus of Dengue Fever.—Ashburn and Craig showed in 
1907. that the virus of this disease exists in the blood of the pa- 
tients and that it is filterable. The disease is probably trans- 
mitted by the mosquito Culex fatigans. Apparently the analogy 
to yellow fever is rather close. 

The Virus of Phlebotomus Fever.—Doerr in .1908 demon- 
strated a filterable virus in the blood of persons suffering from 
the benign three-day fever of Malta and Crete. The disease is 
rather widely distributed in tropical countries. It is transmitted 
by the sand-fly Phlebotomus pa patasii. 

The Virus of Poliomyelitis.—Several investigators, among 
them Flexner and Lewis, demonstrated in 1899 the presenceof a 
filterable virus in the central nervous system of patients suffering 
from infantile paralysis. The virus also occurs in the nasal mucus 
and in the blood. It survives in glycerine for a month, also re- 
sists freezing for weeks, and is rendered inert at 45° to 50° C. in 
30 minutes. It is quickly destroyed by hydrogen peroxide and 
by menthol. 

Flexner and Noguchi? have obtained cultures of the organism 
in ascitic fluid containing sterile tissue and covered with paraffin 
oil, and in this medium rendered solid by admixture of agar. 
The colonies are made up of minute globose bodies 0.15 to 0.30u 
in diameter. Similar bodies have been identified in the nervous - 
tissue from cases of the disease. It seems probable that this 
structure is a living organism and the microbic cause of poliomye- 
litis, especially as inoculation of monkeys with the cultures has 
given rise to the disease. Flexner and his coworkers? regard this 


1 Brit. Journ. Roy. Army Med. Corps, XIV, pp. 1920, Vol. 236-258. 

2 Journ. A. M. A., 1913, V. LX,p. 362. 

3 Amoss, H. L.: Survival of poliomyelitic virus in brain of rabbit, Journ. Exp. 
Med., 1918, 27, p. 443; Smillie, W. G., Cultivation experiments on globoid bodies of 
poliomyelitis, ibid., 1918, 27, P. 319. 


3904 SPECIFIC MICRO-ORGANISMS 


organism as a distinct species and as the microbic cause of the 
disease. Another group! of investigators claim that the globoid 
bodies are diminutive forms of a coccus, which can be grown 
aérobically on blood agar. Their evidence is not considered 
entirely convincing. 

Poliomyelitis or infantile paralysis occurs in epidemics and 
also sporadically, attacking children and young adults. It is 
characterized by digestive disturbance and fever, which may 
be very mild, followed by paralysis of one or more extremities 
as a rule. Death may occur, but recovery with permanent 
paralysis is a frequent result. The mode of transmission is 
unknown. 

The Virus of Measles. — Goldberger and Anderson? in 1911 
reported the successful inoculation of monkeys with the blood, 
nasal and buccal secretions of patients sick with measles and 
also with Berkefeld filtrates. Later investigation? has failed 
to confirm the claims of these authors although several successful 
inoculations of monkeys have been reported.* 

The Virus of Typhus Fever.—Nicolle, Conor and Conseil in 
1910 transmitted typhus fever to monkeys by means of serum 
which had passed through a Berkefeld filter. Ricketts and Wilder 
failed to obtain infective filtrates in their study of Mexican ty- 
phus. Typhus is an acute febrile disease, widely distributed but 
not very prevalent in any locality. Apparently it is not con- 
tagious® but is transmitted from man to man by body lice (Pedi- 
culus vestimenti). Immunity follows recovery. — 

Plotz® and his associates have reported the finding of 
an anaérobic organism in the blood in typhus, Bacillus typhi- 


1Nuzum, J. W.: Journ. Infectious Diseases, 1918, 23, p. 301, p. 309; Rosenow, 
E. C. and others, ibid., 1918, 22, p. 281, p. 313, Pp. 379. 

2 Goldberger and Anderson; Journ. A. M. A., 1911, 57, p. 971. 

3 Sellards and Wentworth: Bull. Johns Hopkins Hops., 1919, 30, p. 57- 

4 Hektoen: Journ. A. M. A., 1919, 72, p. 177. 

5 Wilder: Journ. Infect. Dis., 1911, Vol. IX, p. 9. Ricketts and Wilder: Journ. 
A.M. A., 1910, Vol. LV, pp. 309-311" 

§ Plotz, Olitsky and Baehr: Journ. A. M. A., 1016, 67. D. 1507. 


s THE FILTERABLE MICROBES. 395 


exanthematict, which they consider the cause of typhus. 
Further independent confirmation of their results should be 
awaited. 

The Virus of Small-pox.—The virus of this disease was shown 
to be filterable by Casagrandi in 1908. The vaccine virus, which 
is generally considered to be the same organism, had been pre- 
viously filtered. The organism passes through the coarser Cham- 
berland filters. The virus resists drying for several weeks and 
remains active in glycerine for eight months, but is quickly ren- 
dered inert by bile and by sodium oleate. It is also destroyed by 
heating at 58° C. for 15 minutes. Cell inclusions, which were 
described by Guarnieri in 1892, are considered by some to repre- 
sent forms of the pathogenic agent. 

Small-pox is an acute disease of man characterized by a general 
eruption on the skin, at first papular, then vesicular and pustu- 
lar. It is highly contagious by direct association and by fomites 
and is readily transmitted by placing bits of crust from dried 
pustules on the nasal mucous membrane or on a scratch in the 
skin. Cow-pox is a milder disease which occurs naturally in cows, 
and has also been produced by inoculating calves with small-pox 
virus. An attack of either small-pox or cow-pox is followed by 
immunity to both diseases. Cow-pox in man is a comparatively 
mild disease. Inoculation results in the formation of a single 
pustule, rarely surrounded by secondary vesicles, with slight illness 
for a few days. Edward Jenner in 1798 discovered that cow-pox 
resulting from artificial inoculation(vaccination) confers an immu- 
nity to small-pox. Vaccination is now very generally practised 
in enlightened communities and in such places small-pox is practi- 
cally unknown. The inoculation is best done by making a very 

‘slight superficial linear incision, about 5 mm. long, in the epi- 
dermis and rubbing into it the vaccine virus. The whole pro- 
cedure should result in only 'a faint tinge of blood.. When the 
‘vesicle appears it should be carefully protected from violence. 
A normal vaccination causes little inconvenience and is usually 
completely healed in about 4 weeks after inoculation. Failure 


396 SPECIFIC MICRO-ORGANISMS 


of the inoculation is not a proof of immunity. The vaccination 
should be repeated until it does take. 

The Virus of Chicken Sarcoma.—Rous in 1910 discovered a 
tumor in a chicken which is histologically a typical spindle-cell 
sarcoma and which he has been able to reproduce in other chickens, 
not only by transplantation but also by inoculation of an agent 
which can be separated from the tumor cells! by filtration through 
Berkefeld filters, as well as by inoculation with tumor tissue which 
has been dried and powdered and preserved in the dry condition 

for months. The filterable microbe, or filterable agent as Rous 

conservatively calls it, is rendered inert by heating at 55° C. in 
15 minutes, also by the admixture of chicken bile or saponin. 
Two other sarcomata of the fowl have been shown to be due to a 
filterable agent by the same investigator. 

Our conceptions of the nature of filterable agents is at present 
beginning to become more definite. They are no longer re- 
garded as necessarily beyond the possibility of morphological study 
and there is good reason to hope that the development of improved 
methods of study and their careful application may be able to 
establish not only the important physiological properties of these 
agents but their form and perhaps to some extent their structure 
as well. The beginning already made is full of promise for the 
future.” Filterability does not necessarily mean invisibility. 

In recent years it has become fashionable to accept a filterable 
virus as the cause of an infectious disease in which a visible , 
microbe cannot easily be found, even though the evidence of 
filterability is far from convincing. In some instances the re- 
ported work with filterable viruses has been shown to be unreliable.’ 


1 Rous and Murphy: Journ. Exp. Med., 1913, Vol. XVII, pp. 219-231. Pre- 
vious papers are cited there. ; 

? A number of other diseases have been shown to be caused by filterable agents. 
A brief mention of these together with references to the literature will be found in 
the article by Wolbach: Journ. Med. Rsch., 1612, Vol. XXVII, pp. 1-25. 

’ Bradford, Bashford and Wilson: British Med. Journ., May 17, 1919, 2, pp- 
599-604; Arkwright: A criticism of certain recent claims to have discovered and 
cultivated the filter-passing virus of trench fever and of influenza, Brit. Med. Journ., 
Aug. 23, 1910, 2, D. 233. 


CHAPTER XXVII 
MASTIGOPHORA! 


Herpetomonas Muscez (Domesticz).?—This flagellate proto- 
zoon is commonly found in the intestine of the house fly (Musca 
domestica). The cell body is spindle shaped (Fig. 154) and 15 to 
2su in length. The flagellum is of about 
equal length and contains two stainable 
filaments which terminate near the deeply 
staining blepharoplast situated in the 
anterior part (flagellated 2nd) of the cell. 
From this blepharoplast a delicate thread 
extends in the cytoplasm toward the pos- 
terior end. The nucleus (trophonucleus) 
is at the center of the cell. Multiplication 
takes place by longitudinal division. 

Leptomonas (Herpetomonas) Culicis.* 
In the digestive tract of mosquitoes, 
flagellated organisms occur which bear a 
confusing resemblance to trypanosomes. rig, 154.—Herpelomonas 
They multiply abundantly in the blood musce. «, Normal indi- 

pe ‘ * : - vidual; 6, dividing: form; 
which the insect ingests and are most easily ¢, mode of division of the 
found in the mosquito near the end of De ee 
digestion of a blood meal (48 to 96 hours 
after feeding). The body is 16 to 45m in length and 0.5 to 2u 
in width. Artificial cultures have been obtained in the condensa- 
tion water of blood-agar and these have been purified by streaking 


1 Only a few protozoal forms can be considered and those very briefly. The 
interested student should consult Doflein: Protozoenkunde, III Auflage, Jena, 1911. 

? Prowazek, Arb. Kais. Gesundheitsamt., 1904, Bd. XX, S. 440. 

3 Novy, MacNeal and Torrey: Journ. Inf. Dis., 1907, Vol. IV, p. 223. 


397 


398 A SPECIFIC MICRO-ORGANISMS 


on blood-agar plates. The organism is not known to be capable 
of infecting vertebrates. . . 

Somewhat similar flagellates are found in the alimentary 
tract of various insects, where they may be easily mistaken for 
developmental stages of hematozoa. Trypanosoma (Herpeto- 
monas ) grayi which is found in the tsetse fly Glossina paren 
may be mentioned as another example. 


Fic. 155.—Leptomonas culicis from the digestive tract of ‘a:mosquito. X1500 
(After Novy, MacNeal and Torrey.) i 


Trypanosoma Rotatorium.—This organism is the type species 
of the genus Trypanosoma, as this name was first applied to it by 
Gruby in 1843. It is commonly found in small numbers in the 
_ blood of frogs. The form of the cell varies from that of a slender 
spindle to a very broad and thick structure (Fig. 156). The 
width varies from 5 to 4op and the length from 40 to 804. These 
various forms are probably stages in the growth of the parasite but 
it is not impossible that they represent different species parasitic 
in the same animal. When the larger forms are well stained the 
typical structures of a trypanosome are distinctly evident. The 
large nucleus (trophonucleus) lies near the middle of the body 
and closer to the undulating border. Posterior to it is the smaller 
and more deeply. stained blepharoplast. Close to the latter a 
small clear colorless area is commonly seen. The flagellum 


MASTIGOPHORA 309 


Fic. 156.—Trypanosoma rotatorium in blood of a frog; drawn from a preparation 
stained by Romanowsky method after dry fixation. The smaller form is feebly, 
stained. 


Fic. 157.—Trypanosoma rotatorium. The various forms which occur in arti- 
ficial culture. A, Crithidia form; B, trypanosome form; C, spherical form; D and 
E, club forms; F and G, spirochete forms; H, resting stage; J, resting stage with va- 
cuole and double nucleus. (After Doflein.) 


400 SPECIFIC MICRO-ORGANISMS 


originates near the blepharoplast-and extends along the convex 
border of the cell, which is drawn out into a well-developed thin 
‘undulating membrane, to the anterior end of the cell and beyond 
it as a free flagellum. The posterior tip of the cell is usually 
drawn out to form a slender process. The other border of the 
cell is nearly straight and the cytoplasm near it usually shows 
definite evidence of longitudinal 
striation, indicating the presence 
of elementary muscular structures, 
so-called myonemes. The slender 
form resembles very closely the 
shape of mammalian trypano- 
somes. 

Cultures of Tr. rotatorium were 
first obtained by Lewis and H. U. 
Williams in the condensation fluid 
of slanted blood-agar. Various 
forms of the organism occur in 
the cultures. Many of these are 
doubtless degenerating cells. ‘The 
is, eh Wr iaentionn, et mode of transmission from frog to 

2500. (From Doflein after Minchin.) frog is unknown but it is probably 
, accomplished by means of leeches. 

Trypanosoma Lewisi.—This organism, the common rat 
trypanosome, appears to have been seen as early as 1845, but its 
modern study dates from its rediscovery by Lewis in 1879. It 
occurs in the blood of wild rats throughout the world, from x to 
40 per cent being infected. In the rat the parasite passes through 
a short period, 8 to 14 days, of rapid multiplication, which is 
followed by a period, usually several weeks or months, in which 
the organism persists without evident increase in numbers; 
further multiplication beginning upon transfer to a new host. In 
the adult or resting stage, the trypanosomes are quite uniform, 
1.5 to 24 wide by 27 to 28u in length, including the flagellum 
(Fig. 158). When blood containing these adult forms is injected 


MASTIGOPHORA 401 


into a healthy young rat the multiplication forms of the parasite 
appear after about three days. These forms show a great variety 
of size and shape and they stain more deeply than the adult stage 
(Fig. 159). Numerous dividing parasites are also present, some of 
them showing multiple division with the formation of rosettes. 
The division is longitudinal and essentially unequal, as one cell 
retains the old flagellum while a new one is formed for the other 


bs 


Fic. 159.—Trypanosoma lewisi. Various forms in the blood of a rat six nee after 
inoculation. xX 1125. (After MacNeal.) 

daughter cell. The rosettes arise by successive longitudinal 
divisions, and an unbroken rosette contains one cell with the old 
flagellum larger than the others (Fig. 160). 

The infection is readily transmitted to young rats by the 
injection of blood containing the parasites. Under natural condi- 
' tions transmission is due to insects, especially fleas and lice.! The 


1 Swellengrebel and Strickland: Parasitology, 1910, Vol. III, pp. 360-389. 
26 


402 SPECIFIC MICRO-ORGANISMS 


trypanosomes multiply in the digestive tract of these insects, 
producing various forms, many of them resembling herpetomonas 
and leptomonas. Fleas remain infective for a long time. = 
Cultures of Tr. lewisi were obtained by MacNeal and Novy! 
in 1902-03, in the condensation fluid of inclined blood-agar, and- 
the infection was reproduced by inoculation of these cultures. 


= 


: Fic. 160.—Trypanosoma lewisi. Eight-cell rosette in division. Note the long 
original or parent whip on one of the cells. Several cells show a second flagellum 
growing out preparatory to a further division. x 2250. (After MacNeal.) 


The size and shape of the organism in culture is quite variable. 
The actively dividing forms are usually grouped in rosettes with 
flagella directed centrally, and the cells themselves are pear~ 
shaped or oval. Herpotomonad forms are common. 

The infection with Tr. Jewisi rarely results in death of the rat. 


1 Contributions to Medical Research, dedicated to Victor Clarence Vaughan, 
1903, PP. 549-577. 


MASTIGOPHORA 403 


' 


Other species of animals are not readily infected. Immunity 
follows recovery. Artificial immunity has been produced by 
Novy, Perkins and Chambers! by the injection of a pure culture 
which had been propagated for six ree on artificial media and 
had lost its virulence. 
There are many other relatively harmless trypanosomes 
parasitic in-the blood of various mammals. 
Trypanosoma Brucei.—Bruce in 1895 discovered this organism 


Fic. 161,—The most important trypanosomes parasitic in vertebrates. A, 
Tr. lewisi; B, Tr. evansi (India); C, Tr. evansi (Mauritius); D, Tr. brucei; E, Tr. 
equiperdum; F, Tr. equinum; G, Tr. dimorphon; H, Tr. gambiense. All magnified 
X 1500. (From Doflein after Novy.) : 


in the blood of horses suffering from Nagana, the Tsetse-fly dis- 
ease of Zululand. Pure cultures have been obtained in the con- 
densation fluid of inclined blood-agar by Novy and MacNeal 
and the injection of pure cultures into animals produces the dis- 
ease and death. 

Tr. brucei is 1.5 to 54 wide and 25 to 35u long, including the 


} Journ. Inf. Dis., 1912, Vol. XI, pp. 411-426. 


404 SPECIFIC MICRO-ORGANISMS 


| flagellum. The nucleus lies near the center of the cell. It is oval 
or somewhat irregular in outline and usually occupies the whole 
width of the cell. Near the blunt posterior end of the cell is a 


A 


Fic. 162.—Glossina morsitans. A, Magnified. (After Doflein.) B, Sketch showing 
natural size. (From Doflein after Blanchard.) 
spherical granule, the blepharoplast. Near this the flagellum 
_ originates and it extends forward along the convex border of the . 
cell, which is drawn out into a thin undulating membrane, and 
B 


Fic. 163.—Glossina morsitans; lateral view of the resting fly. A, Before feeding. 

B, After sucking blood. (From Doflein after Austen.) : 
extends beyond the anterior end of the cell as a free flagellum. 
The cytoplasm anterior to the nucleus often contains many coarse 
granules. -The general shape of the trypanosome as seen in the 


MASTIGOPHORA 405 


plood of the infected animal is fairly uniform. There is, however, 


considerable variety in size, internal structure and staining 
properties. Multiplication takes place by unequal longitudinal 
division, much the same as in Tr. lewisi, but the dividing cell has 
the same general form as the others and multiple division figures 
are less common. The larger cells are usually in process of divi- 
sion. Trypanosomes with feebly staining cytoplasm and others 
with very abundant coarse granules also occur. The former are 
probably degenerating and disintegrating cells. 

Tr. brucei is taken up by the blood-sucking tsetse fly, Glessina 
morsttans and in about 5 per cent of these it multiplies in the 
alimentary canal and penetrates into the body cavity, causing a 
generalized infection of the fly. After about three or four weeks 
the salivary glands are invaded and the fly is then able to infect 
other animals by biting them, and it remains infective for a long 
time, probably as long as it lives. Other insects may possibly 
serve to transmit the parasite. The infection,is also readily 
transmitted from animal to animal by the injection of infected 
blood. 

Cultures are obtained with some difficulty, but most readily 
by inoculating inclined blood-agar,! 2:1, and incubating at 28° C. 
The primary cultures should not be transplanted until they are 
about three weeks old, and they usually fail to infect animals if 


injected into them. The virulence is regained in the subcultufes. _ 


Culture filtrates are not toxic. The poison of trypanosomes 
seems to be set free as a result of their disintegration in the body 
fluids.? 

Nagana occurs naturally in a great variety of the quadrupeds 
and is usually fatal. Man is not susceptible. Mice and rats 
die in 6 to 14 days after inoculation. Guinea-pigs may show one 
or more relapses, the disease lasting for two to ten weeks. 


1 The agar employed should contain the extractives of 125 grams of meat, 1 
grams pepton, 5 grams salt and 25 grams of agar in 1000 C.C. It is liquefied, cooled to 
50° C. and mixed with twice its volume of warm defibrinated rabbit’s blood and then 
allowed to solidify in an inclined position. 

2 MacNeal: Journ. Inf. Dis., 1904, Vol. I, p. 537. 


7 


» 


i 


HOO. « _ SPECIFIC MICRO-ORGANISMS 


Diagnosis may be made by microscopic examination of the 
blood when the parasites are numerous. At other times it is well 
to inject 5 to ro c.c of blood into a white rat. The distinction 
of Tr. brucei from other species of trypanosomes causing similar 
diseases is not easy and may require prolonged study. 

Immunity. of susceptible animals has not yet been achieved, 
but inoculation with attenuated cultures produces a relative 
immunity in small laboratory animals.! 

Trypanosoma Evansi.—This organism was diceverel: by 
Griffith Evans in 1880 in the blood of horses and various other 


Fic. 164.—Trypanosoma equiperdum. Blood of an inoculated rat. A, after four 
days; B, after eight days. (After Doflein.) 


animals suffering from the disease known in India as Surra. . The 
trypanosome resembles Tr. brucei in most respects but is recog- 
nized as a distinct species. Surra is apparently transmitted by 
various flies, Tabanide, Stomoxys, and also by fleas. 

Trypanosoma equiperdum was found by Rouget in 1896 in 
the blood of horses suffering from dourine. The infection is 
transmitted by coitus and probably also in other ways. Dourine - 
occurs in southern Europe and northern Africa. A few cases 
have been observed in Canada and in the United States. Small 
laboratory animals are susceptible to inoculation. 


Novy, Perkins and Chambers: Journ. Inf. Dis., 1912, Vol. XI, pp. 411426. 


MASTIGOPHORA 407 


Trypanosoma Equinum.—Elmassian in 1901 observed this 
organism in the blood of horses suffering from Mal de Caderas 
in South America. It possesses a very minute blepharoplast, a 
morphological character which distinguishes it from most other 
trypanosomes. Small laboratory animals are susceptible. = 

Several other species of trypanosomes have been described, 
which cause fatal diseases in quadrupeds. Most of these have 
been found in Africa. 

Trypanosoma Gambiense.—Dutton and Todd in 1901 .ob- 
served this organism in the blood of an Englishman in Gambia. 
The parasite had been previously seen by Forde. The disease, 
which resulted in death after two years, was called trypanosoma 
fever. Castellani in 1903 observed trypanosomes in the cerebro- 
spinal fluid of patients suffering from sleeping sickness in Uganda. 
This organism is now known to be the same as the Tr, gambiense 
of Dutton, and sleeping sickness is recognized as the terminal 
stage of trypanosoma fever. 

Tr. gambiense is very similar in form to Tr. brucei but the 
posterior end is on the average somewhat more pointed. The 
length varies between 15 and 30 and the width from 1 to 3u. The 
significance of the different forms found in the blood is not defi- 
nitely known. Multiplication takes place in the same way as 
in Tr. brucei. In the tsetse fly, Glossina palpalis, the trypano- 
somes slowly disintegrate and disappear during the first four days 
after the infected blood is ingested, and in most of the flies this 
results in extermination of the trypanosomes. In 5 to Io per 
cent of the flies the parasites are not completely destroyed, but 
the early diminution in their number is followed by an abundant 
multiplication of the trypanosomes in the stomach and intestine of 
the insect. After 18 to 53 days these flies become capable of 
__ infecting new animals by their’ bite and remain infectious for a 
very long time. The parasites are found in the salivary glands? 
when the ‘fly becomes capable of causing the disease. A great 


1 Bruce, Hamerton, Bateman and Mackie: Proc. Royal Soc., 1911, Ser. B, 
Vol. LXXXIII, pp. 338-3445 Pp. 345-348; Pp. 513-527. 


408 SPECIFIC MICRO-ORGANISMS 


diversity of form is observed in the trypanosomes within the fly but 
the significance of the different types is not yet fully understood. 

Many of the mammals are susceptible to inoculation with 
Tr. gambiense. White rats usually relapse 2 or 3 times before 
finally succumbing to the infection, whereas they usually die 
within 2 weeks when inoculated with Tr. brucei. The virulence 
of the organism is somewhat variable. 

Attempts to cultivate Tr. gambiense in artificial media have 
not been fully successful. It has been possible to obtain multipli- 


Fic. 16 5.—Glossina palpalis in natural resting position, and with wings outstretched. 
; (After Doflein.) 


cation of the organisms and to keep them alive for several weeks 
on blood-agar but such cultures are not virulent and cannot be 
kept up indefinitely.? 

Human trypanosomiasis is a most important and widespread 
disease in equatorial Africa. Symptoms appear long after the 
infection has taken place. The disease manifests itself in two 
forms, the trypanosoma fever and the sleeping sickness. Try- 
panosoma fever is an irregularly remittent fever lasting for several 
days at each attack, accompanied by a macular eruption, and 


‘Thomson and Sinton: Annals of Trop. Med. and Parasitol., 1912, Vol. VI, 
PP- 331-356. 


MASTLGORMORA 409 


always associated with a general enlargement of the lymph nodes. 
The trypanosomes are numerous in the blood during the febrile 
period and become very scarce during the intermissions. The 
fever leads to emaciation and death, sometimes without inducing 
the terminal coma and sometimes with the production of typical 
sleeping sickness. The sleeping sickness is characterized by pro- 
longed coma and progressive emaciation. At intervals the 
patient may be aroused and given nourishment, but eventually 
this is no longer possible. At this stage the trypanosomes are 
present in the cerebrospinal fluid. Bacterial infection of the 


Fic. 166.—Trypanosoma avium in the blood of common wild birds. X 1500. 
(After Novy and MacNeal.) 


meninges often takes place as a terminal event. It is conserva- 
tively estimated that roo,ooo natives have died of trypanosomiasis 
in Africa from 1900 to 1910. There have been several cases in 
Europeans. Recovery seems to be rather uncommon but does 
occur. tae . 

Trypanosoma Rhodesiense.—Stephens and Fantham! have 
studied a case of human trypanosomiasis contracted in north- 
eastern Rhodesia, where Glossina palpalis does not occur. The 
parasite differs somewhat from Tr. gambiense and is regarded by 


1 Proc. Royal Soc., 1910, Ser. B, Vol. LX XXIII, pp. 28-33. 


410 : SPECIFIC MICRO-ORGANISMS 


these authors as a distinct species. It seems to be transmitted 
by Glossina morsitans.' 

Trypanosoma Avium.—Trypanosomes were probably seen in 
the blood of birds by earlier investigators, but the first accurate 
description of such observations is that of Danilewsky in 1885. 


Fic) 167.—Trypanosoma avium in culture on blood agar; X 1500. (After Novy 


and MacNeal.) 
Infection with trypanosomes is very common in the ordinary 
wild birds. Novy and Mac Neal? examined 431 American birds 
representing 40 common species and found trypanosomes in 38 
individuals, representing 16 species. The indicated prevalence. 


1 Kinghorn and Yorke: Annals of Trop. Med. and Parasitol., 1912, Vol. VI, 
pp. 269-285. Kinghorn, Yorke and Lloyd: ibid., 1912, Vol. VI, pp. 495-503. 
* Journ. Infect. Dis., 1905, Vol. II, pp. 256-308. 


MASTIGOPHORA AIL 


of the infection, 8.8 per cent, is doubtless far below the actual 
percentage, as many of the birds were not tested by the cultural 
method. There are doubtless several species of bird trypanosomes 
but. the most common form is Tr. avium. The length varies 
from 25 to 7ou and the width from 4 to 7. 

Cultures are easily obtained by transferring the infected blood 
to tubes of blood-agar and incubating at 25° to 30° C. The pro- 
tozoa grow abundantly and, by weekly transfers, may be kept 
under cultivation without special difficulty for an indefinite period. 
Injection of cultures into birds is only rarely followed by appear- 
ance of trypanosomes in the blood. 

The parasites persist in the blood.of the birds for many months 
and probably for years. They seem to be comparatively harmless. 
The mode of transmission from bird to bird is unknown. 

Trypanosoma avium is a form of considerable importance in 
the study of systematic protozodlogy because of the confusion of’ 
trypanosomes and hemocytozoa by Schaudinn! in 1904, who 
regarded Tr. avium as merely an extracellular form of Hemopro- 
teus noctue (danilewskyi?) (see page 433). This misconception, 
together with the analogous assumption of similar relationship 
between spirochetes of birds and the leukocytozoén of Ziemann, 
Hemoproteus ziemanni, made by Schaudinn at the same time, 
has exercised a profound influence upon the course of investiga- 
tion in the groups of spirochetes, trypanosomes and hemocytozoa. 

Schizotrypanum Cruzi— Chagas discovered this organism 
in 1907. It occurs in the blood in the Brazilian human trypano- 
somiasis called coreotrypanosis. Multiplication takes place 
within endothelial cells, lymphocytes and other cells in the paren- 
chymatous organs, and especially in the interior of muscle cells in 
the heart and skeletal muscles.2. The dividing parasites are with- 
out flagella and resemble the intracellular forms of Leishmania. 
From these cysts the parasites escape into the blood, where they 


1 Arb. a. d. Kais. Gesundheitsamte, 1904, Vol. XX, pp. 387-430. 
*Vianna: Memorias do Instituto Oswaldo Cruz, 1911, Vol. III, pp. 276-293. 
Abstract in Sleeping Sickness Bull., 1912, Vol. IV, pp. 288-293. 


. 412 SPECIFIC MICRO-ORGANISMS 


4 ’ RL, SHEPPARD. 


_ Fic. 168.—Schizotrypanum cruzi developing in the tissues of the guinea-pig. 
1. Cross-section of a striated muscle fiber containing Schioztrypanum cruzi: Note 
dividing forms. 2. Section of brain showing a Schizotrypanum cyst within a 
neuroglia cell, containing chiefly flagellated forms. 3. Section through the supra- 
renal capsule, fascicular zone. 4. Section of brain showing a neuroglia cell filled 
with round forms of Schizoirypanum. (From Low, in Sleeping Sickness Bulletin, 
after Vianna.) 


‘ 


MASTIGOPHORA 413 


are found as trypanosomes in the blood plasma. Slender and 
thick forms occur here, the difference probably depending upon 
the age of the parasites. 

Monkeys, rats, mice, young guinea-pigs and many other 
mammals are suceptible to inoculation. The infection is tran- 
mitted by a bug, Conorhinus megistus, in which the protozoén 
develops abundantly. The bedbug, Culex lectularius, also is 
capable of transmitting the disease. . 

Cultures are readily obtained 
on blood-agar and Chagas was able 
to infect animals with such cultures. 

Leishmania Donovani.—Lav- 
eran and Mesnil in 1903 described 
this protozoén which occurs inside 
cells in various parts of the body, fee a pre 
but is especially abundant in the after Chagas.) 
spleen and liver, in the disease 
known in India as Kala-Azar or tropical splenomegaly. The 
organism is oval, 2 to 4u in diameter, finely granular and some- 
times vacuolated. In the interior there is a large rounded nucleus 
and a smaller oval or rod-shaped blepharoplast, near which a third 
very slender short thread may usually be recognized as the rudi- 
ment of the undeveloped flagellum. These structures are doubled 
in the division stages. Multiple division also occurs. In the cir- 
culating blood the organism is found within lymphocytes and poly- 
nuclear leukocytes. Many of them may be found in a single cell. 

Cultures are readily obtained by inoculating fluid (citrated) 
blood with blood or with spleen juice containing the parasites, or 
by inoculating the usual blood-agar. In artificial culture the 
‘cell elongates, the rudimentary whip extends into a true flagellum 
and the organism assumes the appearance of a typical leptomonas 
(herpetomonas). Little difficulty is experienced in keeping the 
cultures alive and flourishing. 

The parasite has been supposed to be transmitted from man 
to man by bugs of the genus Cimex, but this hypothesis has been 


AI4 SPECIFIC MICRO-ORGANISMS 


rendered very uncertain by the work of Wenyon' and the Ser- 


Fic. 170.—Conorhinus megistus, the insect carrier of Schizotrypanum cruzi. (From 
Doflein after Chagas.) 


Fic. 171.—Leishmania donovani in the juice obtained by puncture of the spleen in 
kala-azar. (From Doflein after Donovan.) 

gents.” The latter investigators were able to effect experi- 

mental transmission by means of the dog flea, Ctenocephalus canis. 


1 Journ. Lond. Sch. Trop. Med., 1912, Vol. II, pp. 13-26. 
2Sergent (Edm. & Et.), L’Heritier and Lemaire, Bull. Soc. Path, Exot., 1912, 
Vol. V, pp. 595-597. 


_ MASTIGOPHORA 


415 


Kala-Azar is endemic in tropical Asia and northeast Africa, 
where it occurs among the poorer class of people, living in squalor. 


It is characterized by irregular fever, weak- 
ness and cachexia and especially by enor- 
mous enlargement of the spleen, often of 
the liver also. It is frequently fatal. 
Dogs and monkeys are susceptible to in- 
oculation. ; 
Leishmania Tropica.—This organism 
was first accurately described by J. H. 
Wright,! who found it in great abundance 
in the lesion known as Aleppo boil, Delhi 
boil or tropical ulcer. The parasites occur 
within the endothelial cells within the 


ene 
| “4 


Fic. 172.— Leishmania 
donovani, various forms 
observed in artificial cul- 
ture. (From Doflein after 
Chatterjee.) 


lesion and are very 


Fic. 173.—Leishmania tropica. Smear from a Delhi boil. 1500. (After 


J. H. Wright.) 


numerous. Leishmania tropica resembles L. donovani very 


closely except in its pathogenic properties. 
c 1 Journ, Med. Rsch., 1903, Vol. X, pp. 472-482. 


Cultures on blood- 


416. SPECIFIC MICRO-ORGANISMS 


¥ 


agar have been obtained by Nicolle and are easily propagated 
at 22” C. Dogs and monkeys are susceptible to inoculation and 
the human disease is probably contracted from dogs through © 
the agency of insects. The disease is relatively benign and recov- 
ery is followed by prolonged immunity. Inoculation has been 
practised in man in order to produce immunity. 


Fic. 174.—Leishmania tropica, Fic. 175.—Trypanoplasma  cy- 
forms observed in cultures. (From print. Bl, Blepharoplast;, N, nu- 
‘Doflein after Nicolle.) cleus. XX 2000. (After Doflein.) 


Leishmania Infantum.—Nicolle in 1908 observed this organ- 
ism in the spleen, liver and bone marrow of children dying from 
splenomegaly in northern Africa. The disease resembles Kala- 
Azar in all respects except that the patients are all very young. 
Dogs are. naturally infected with this parasite and are probably 
the source of the human disease. Cultures on blood-agar are 
readily obtained and kept up indefinitely without special difficulty. 


MASTIGOPHORA 417 


Trypanoplasma Borreli—Laveran and Mesnil in 1901 de- 
scribed this protozoén, which occurs in the blood of various species 
of fish. It resembles a trypanosome somewhat, but the blepharo- 
plast is relatively large and from it two flagella originate, one 
extending forward immediately as a free whip while the other runs 

2. 4 5 
along the convex border, ensheathed in an undulating membrane, 
* . 

and extends at the posterior end as 
a free flagellum. Longitudinal division 
takes place in the circulating blood. 
Transmission seems to be accomplished 
by means of leeches. T. cyprini and 
T. guerneit seem to be identical with 
T. borreli, but they may prove to be 
distinct species. 


= € 
Fic. 176.—Bodo lacerte. u, Fic. 177.—Trichomonas hominis from the 
Sketched from life; b, drawn from mouth. (From Dofiein after Prowazek.) 
a stained preparation. (From 
Doflein after Hartmann and 
Prowazek.) ~ 


Bodo Lacerte.—In the cloaca of various lizards’ a flagellate 
is almost constantly found. It is 2 to 4u wide and 6 to 12.5u 
long, lance-shaped and twisted at the posterior (pointed) end. 
The nucleus is near the anterior end. At its side is a granule 
resembling a blepharoplast and from this a thread extends to 


the anterior cell end of the cell, where it gives rise to two flagella. 
27 


SPECIFIC MICRO-ORGANISMS 


A B D 
Fic. 178.—Lamblia intestinalis. A,” Ventral ‘aspect; B, lateral view; C, in posi- 
tion on epithelium; D, the same enlarged. (From Doflein after Grassi‘and Schewia- 


kof.) 
GE 


Fic. 179.—Trimastigameba philippinensis. A, Early stage of division of the 
nucleus. The polar caps are still united by a bridge. The equatorial plate has 
formed. B, Ordinary cyst. C, Vegetative form showing the nucleus and a second 
chromatin granule (split off from it?). D, Flagellated form showing remains of the 
rhizoplast between the nucleus and the basal granules. E, Flagellated form with 


pseudopodia. (After Whitmore.) 


MASTIGOPHORA 419 


Trichomonas Hominis.—Davaine observed this parasite in 
1854. It is common in the human digestive tract, especially in 
the stomach in anacidity and in the intestine in chronic digestive 
disturbances. The organism is 3 to 4u wide and 4 to 15y long, 
pear-shaped and provided with three free flagella, and a fourth 
thread which passes around one side of the cell in the margin of 
the undulating membrane. The parasite seems to be a harmless 
commensal, as a rule, but it may possibly bear some causal rela- 
tion to diarrhea in some cases. Animals have not been success- 
fully inoculated with it. Tr. vaginalis is very similar. It grows 
in the acid vaginal mucus. Other trichomonad forms occur in 
the intestines of animals, particularly in mice, in frogs and in 
lizards. 

Lamblia Intestinalis—The cell has the form of a turnip with 
a wide and deep excavation in front near the anterior rounded 
end, forming a suction cup. The body is bilaterally symmetrical. 
The length is 10 to 21m and the width 5 to 124. There are eight 
flagella, each from 9 to 14u- long. The mode of multiplication is 
not fully known. Resistant cysts are formed, probably after 
sexual union of two individuals, and these escape with the feces 
and lead to the infection of new hosts. Lamblia lives in the duo- 
denum and jejunum of man and many other mammals. It ap- 
pears to be relatively harmless in most cases but the possibility 
that it may be a cause of digestive disturbance must be con- 
sidered. It is often present in chronic dysenteries. 

Mastigameeba Aspera.—This is a saprophytic form, described 
by Schulze, which possesses a single flagellum, but is also capable 
of extending finger-like projections of its cytoplasm, pseudopodia, 
just as an ameba does. Whitmore! has described a somewhat 
similar saprophyte, Trimastigameba philippinensis, which is at 
times ameboid without flagella and at other times possesses three 
or possibly four whips. It divides and encysts like an ameba. 
The organism is readily cultivated on the alkaline agar of Mus- 
grave and Klegg. 

1 Archiv f. Protistenkunde, 1911, Bd. XXIII, S. 81-95. 


CHAPTER XXVIII 
RHIZOPODA: 


Amoeba Proteus.—This large saprophytic ameba may ‘be 
considered as an example of the numerous species of free-living 
amebe,: the classification and identification of which is still in 
hopeless confusion. The organism is widely distributed in stag- 
nant water and is easily cultivated in the laboratory in not too 
foul infusions containing bacteria and alge. The cell is 50 to 


Fic. 180.—A, Ameba proteus engulfing a clump of small alge (Na). Cv, con- 
tractile vacuole; N, nucleus. 3B, Newly encysted ameba showing nuclear fragments; 
cy, cyst wall; x, nucleus; R, reserve food substance. C, Cyst containing many young 
amebz beginning to escape; cy, cyst wall; k, young amebe. (After Doflein.) 

500% across, often possesses numerous thick, blunt pseudopodia: 

The ectoplasm and endoplasm appear distinctly different, the 
latter being filled with granules, crystals, vacuoles and food parti- 
cles, such as alge and bacterial cells, and possessing a contractile 
vacuole. The nucelus is lentil-shaped and the chromatin within 
it has a very typical arrangement in a central plate surrounded 
by a network on which the peripheral chromatin is symmetrically 

: 420 


RHIZOPODA 421 


placed. Binary division with mitosis of the nucleus seems to be 
. the common mode of multiplication. Multiple division also 
occurs in the vegetative state. The resistant stage (cyst) is char- 
acterized, by a thick, firm wall of several layers, within which the 
nucleus divides into 200 or more daughter nuclei. Each of these 
becomes surrounded by a little cytoplasm and, when the cyst. 
bursts, wanders out as a young ameba. The life history is in- 
completely known. 
Cultures of saprophytic amebe are readily obtained upon 
agar plates. The medium contains agar 0.5 gram, tap water 


/ Fic, 181.—Endameba coli. a, Free ameba; b, ripe cyst with eight nuclei. (From 
Doflein after Hartmann.) 


go c.c., ordinary nutrient broth 1o c.c. Cultures are incubated 
at 25° C. Williams! has succeeded in obtaining pure cultures 
free from bacteria, at 36° C. by employing agar smeared with. 
naturally sterile brain substance. 

Endamceba Coli.—Loesch? in 1875 observed amebe in the 
human large intestine in gastro-intestinal disturbance. The 
organism is very common in the human intestine, being found in 
Io to 60 per cent of persons without digestive disturbances, 
when the examination is thorough. 

The cell in the vegetative stage is variable in shape and size, 


1 Journ. Med. Rsch., 1911, Vol. XXV, pp. 263-283. 
? Virchow’s Archiv, 1875, Bd. LXV, S. 196-211. 


422 SPECIFIC MICRO-ORGANISMS 


the diameter measuring 10 to 7ou. The protoplasm is slightly 
granular and shows distinctly an alveolar structure. The dis- 
tinction between ectoplasm and endoplasm is apparent only in 
the pseudopodia. There is no contractile vacuole. Food sub- 
stance is present in the cytoplasm, bits of vegetable material, 
bacteria and, rarely, red blood cells. The nucleus is round, ve- 
sicular and enclosed in a nuclear membrane. In its center is a 
relatively large mass of chromatin and there are numerous smaller 
masses of chromatin at the periphery beneath the nuclear mem- 
-brane. Multiplication in the vegetative stage takes place by 
binary division as a rule, but multiple division preceded by re- 
peated division of the nucleus also occurs. 

E. coli discharges all food material from its cytoplasm before 
encystment so that the cell is clear and the nucleus plainly visible. 
A large vacuole in the cytoplasm usually makes its appearance 
and is present during the first and second division of the nucleus 
in the cyst. It is large in those cysts in which much chromatin 
escapes from the nuclei into the cytoplasm as chromidia, and it 
usually disappears when the four, nuclei have been formed. A 
further division of the nuclei gives rise to eight and this is the 
usual number present in the fully developed cyst of E. coli, al- 
though rarely ten or even sixteen nuclei may be observed. The 
self-fertilization, autogamy, described by Schaudinn as occurring 
early in encystment has not been observed by Hartmann, and 
its actual occurrence seems questionable. The developed cyst 
with eight nuclei is about 154 in diameter and is considered to be 
definitely characteristic of this species. 

E. coli is generally regarded as a harmless commensal in the 
human intestine. It is however impossible to exclude the possi- 
bility that it may contribute to the aggravation of pathological 
conditions present in the digestive tract. (Compare with Bacillus 
coli.) Its common occurrence in healthy men speaks against 
its possessing any very specific and powerful pathogenic property. 


1 Hartmann and Whitmore: Archiv. f. Protistenkunde, 1912, Bd. XXIV, S. 
182-194. 


RHIZOPODA 423 


Endameeba Dysenterie.—It is evident that the dysentery 
ameba was observed by Loesch in 1875, but a clear distinction 
between the harmless and the pathogenic forms of enteric amebe 


Fic. 182.—Endameba dysenterie. The same living individual drawn at brief inter- 
vals while moving. (From Doflein’ after Hartmann.) 


was not made until 1903. It is yet not certain whether one or 
several species are included in the dysenteric amebe. Thename, 


Fic. 183.—Endameba dysenterie. a, Vegetative cell containing a red blood cell 
(near upper end). 1300. band c, Drawings of nuclei showing stages of the so- 
called cyclical changes. X2600. (From Doflein after Hartmann.) 


Endameba dysenteric is synonymous with E. tetragena and E. 
histolytica. The organism occurs in the intestine and in the stools 
of persons suffering from amebic dysentery and very seldom in 


424 SPECIFIC MICRO-ORGANISMS 


other individuals. The cell is 8 to 6ou in diameter. The ecto- 
plasm is distinctly differentiated from the endoplasm even when 
the cell is motionless, and the lobose pseudopodia are made up 
entirely of the stiff highly refractive ectoplasm. The endoplasm | 
contains food material consisting of bacteria, cell fragments and 
red blood cells. The nucleus is very distinctly visible in the 
living ameba. It is spherical and surrounded by a thick doubly.. 
contoured nuclear membrane. The chromatin is usually dis- 
tributed just beneath the nuclear membrane. 
in largest amount and in the center there is a 
karyosome with definite centriole. The vege- 
tative multiplication takes place by division into 
two daughter cells. Multiple division seems not 
to occur. 


Fic. 184.—Enda- oe 
maba dysenteriae. Cyst formation is rarely observed. The cysts 


Mature cyst con- * i s 
taidtae four mudi, ME most likely to be found when the stool be 


anda massof chro- comes formed in convalescence from an attack of 

midial substance. . . 

(After Hartmann, dysentery and they may then be very numer- 
‘ous. The mature cyst contains four nuclei,. 

and frequently contains also one or more large masses of chromidial 

substance which stain black with iron hematoxylin. 

The forms of the organism commonly observed in the feces of - 
dysentery are either the active vegetative cells! or degenerating 
forms, and the latter may lead to confusion unless their true nature 

_ is recognized. 

The belief that amebe bear a causal relation to dysentery is 
based upon the fact that certain types of amebe, E. dysenterie 
(E. histolytica) are found in the stools, as a rule, only in cases of 
dysentery; further, that these cases of dysentery, in which these 
amebz occur, are characterized by definite clinical signs and 
typical anatomical changes in the intestine; and that these amebe 
are found penetrating deeply into the mucosa of the intestine, 
and it is possible to produce ulcerative enteritis in experimental 


1 Hartmann: Arch. f. Protistenkunde, 1912, Bd. XXIV, S. 163-181. 


RHIZOPODA 425 


animals by injecting feces containing amebe into the rectum or. 
by feeding fecal material containing cysts; and further, the fact: 
that abscesses occur in the liver in amebic dysentery, in which the 
amebe are present and in which it has been impossible to demon- 
strate the presence of bacteria. The causal relation seems highly 
probable, but it must be recognized that the evidence is very in- 
conclusive and admits of other possible explanations. Even 
the relationships of the various forms seen in the microscopic 
preparations require a certain amount of speculation for their 
determination, and the possibility of error, even by the experienced 
protozodlogist, must be recognized and has been’ well illustrated 
by the divergent views of Schaudinh and of Hartmann in study- 
ing the same slides. Greater certainty ,would doubtless be 
derived from the study of artificial cultures if such could be made 
available. 

Numerous cultures of amebe have been obtained from the 
stools of cases of dysentery, and some from the pus of amebic 
abscesses of the liver, the growth taking place on agar in the pres- 
ence of a single species of bacteria. With these cultures it has 
been possible to cause enteritis in monkeys. Such cultures have 
also been grown at 37° C. by A. W. Williams! in pure culture on 
agar streaked with brain substance and with blood, and in these 
cultures she finds that the am2be approach in their structure 
the typical endamebe, not only in nuclear structure and cyst 
formation, but also in the utilization of red blood cells as food. 
Whitmore? has carefully studied a number of cultures of amebe 
obtained from cases of dysentery, one of them from a liver abscess, 
and has concluded that in every instance the amebz were free- 

_living saprophytic forms belonging to the genus Ameba and not 
in any case parasitic species. 


1Soc. Amer. Bact., New York Meeting, Jan. 2, 1913. Science 1913, Vol. 
XXXVIII, p. 451; Williams, A. W., and Calkins, G. N., Journ. Med. Rsch., 1913, 
Vol. XXIX, pp. 43-56. 

* Archiv f. Protistenkunde, 1911, Bd. XXIII, S. 71~80; ibid., pp. 81-95. 


426 SPECIFIC MICRO-ORGANISMS 


Other Rhizopoda.—The remaining orders of the Rhizopoda, 
namely Helizoa, Foraminifera, Radiolaria and Mycetozoa contain 
no parasitic forms of great importance to human pathology. 
Plasmodium brassice which causes tumors on the roots of the 
cauliflower plant is of some interest.! 


1See Doflein, Protozoenkunde, 1011, S. 672-678. 


CHAPTER XXIX 
SPOROZOA 


Cyclospora Caryolytica.—Schaudinn in 1902 discovered this 
organism, which lives as a parasite in the nuclei of epithelial cells 
of the intestinal mucosa in the common mole. It is ingested in 
the form of spores, from which the slender young sporozoites 
escape in the intestine and penetrate the nuclei of epithelial cells. 
Here the parasite becomes rounded and enlarges; becoming 
quickly differentiated into either the male or female type. The 
former type of parasite has numerous refractive granules in its 


Fic. 185.—Cyclospora caryolytica. A, Male cells within the nucleus of the host . 


cell, B and C, Reproduction by multiple division with final rupture of the host 
nucleus in (C). (From Doflein after Schaudinn.) 


cytoplasm, while the female type has a clear cytoplasm. The 
parasites grow rapidly and segment after 4 to 8 hours, the females 
earlier than the males, and the cells resulting from this segmenta- 
tion, so-called merozoites or agametes, penetrate new nuclei and 
go through the same development. Four to five days after in- 
fection of the mole, the parasites suddenly cease their asexual 
multiplication. The male parasites, microgametocytes, after 
rapid multiplication of nuclei, give rise to numerous microgametes 
427 


428 SPECIFIC MICRO-ORGANISMS 


provided with two flagella. The female cells, macrogametocytes, 
enlarge slowly and produce numerous yolk-like granules in their 


Fic. 186.—Cyclospora caryolytica. A, Female cell (agamete) within the host 
nucleus. Band C, Multiple division. D, A free young female agamete. (From 
Doflein after Schaudinn.) ; 
cytoplasm. The nucleus undergoes two reduction (maturation) 


- divisions, and one daughter nucleus remains while the others 


Fic. 187.—Cyclospora caryolytica. A, Fertilization. B, Fertilized cell. C, Fer- 
tilized cell (odcyst) with cyst wall. D, E, F and G, Division of the cyst contents to 
form two spores, each containing two sporozoits. H, Escape of the sporozoits. 
(From Doflein after Schaudinn.) ‘ 


_ disintegrate. Several microgametes penetrate the matured macro- 
gamete and one of them unites with the nucleus. A cyst wall 


SPOROZOA. 429 


forms about the fertilized cell and within this the cell divides, 
into two and later into four embryo parasites, which are enclosed 
in pairsin two spores within the cyst. This escapes with the 
feces of the mole and serves to infect a new host. 

The invasion of the epithelium produces a severe diarrhea in 
the mole often resulting in death. If the animal survives for 
five days, until after the spores are formed, it 
then usually recovers. 

Eimeria Stiedz (Coccidium Cuniculi).—This 
very common parasite of the rabbit was first de- 
scribed by Lindemann in 1865. It lives and grows [fg 
within the epithelial cells of the small intestine, of 
the bile passages and of the liver of rabbits suffer- 
ing from coccidiosis, and its odcysts are found in 
the intestinal contents and in the feces of such 
animals. The odcyst is an elongated oval, vari- 
able in width from 11 to 28 and in length from 


Fic. 188.—Ei- 
meria steide. 
Oécyst containing 
four. spores, in 


24 to4gu. It contains, when fully developed, four 
spores, each of which contains two embryo para-, 
sites or sporozoits. These gain entrance to the 
intestine of a new host along ‘with the food and the 


each of which two 
sporozoits are de- 
veloping. The 
micropyle is be- 
low. (From Do- 
flein after Meiz-. 


pancreatic digestion makes .an opening at one end a 


where the wall is exceedingly thin, the micropyle, and through this 
opening the wedge-shaped sporozoits escape. They penetrate epi- 
thelial cells, in which the parasite becomes rounded and grows toa 
diameter of 20 to sou, destroying the host cell. The nucleus 
divides many times and after it the cytoplasm, so as to form numer- 
ous spindle-shaped young cells, merozoits or agametes, which 
penetrate new epithelial cells and pass through the same cycle. 
* This cycle of asexual multiplication, schizogony, is repeated many 
‘times and may lead to extensive destruction of intestinal mucosa, ~ 
of the epithelium of the bile ducts and of liver substance. Some of 
the growing parasites become differentiated into sexual elements. 
The female cell, macrogametocyte, accumulates numerous large 
granules in its cytoplasm, and when full-grown the chromatin 


430 SPECIFIC MICRO-ORGANISMS 


of the nucleus is reduced by expulsion of the karyosome. The 
matured cell, macrogamete, is then ready for union with the 
‘microgamete. The growing cell destined to give rise to the male 
sexual elements attains a large size and possesses a pale cytoplasm... 
It is called the microgametocyte. Its nucleus divides many 
times, the small nuclei accumulate near the surface of the ‘cell 
and each escapes with a small portion of protoplasm as a slender 
motile microgamete. The penetration of one of these into the 
macrogamete produces the fertilized odcyst, which forms a thick 
wall about itself and escapes to the external world. Here, the 


Fic. 189.—Eimeria steide. a, Young agamete (merozoit). 0, Epithelial cell 
invaded by three young agametes. c, d, and e, Stages in the multiple division of the 
agamete. jf, Young macrogametocyte. g, Full-grown macrogametocyte. (From 
Doflein after Hartmann.) ; 


fertilized cell divides to form eight cells, sporozoits, which are 
enclosed within four oval spores (two in each) within the wall of 
the odcyst. If this cyst is ingested by another rabbit the cycle 
of development starts anew. ; 

Coccidiosis is a very common disease in rabbits. The animal 
suffers from severe diarrhea and loss of appetite, and becomes 
emaciated. Young rabbits often die of the disease. Diagnosis 
is readily made by finding the odcysts in the feces. Children 
have been found to be infected with this organism. Cattle, 
horses, sheep and swine are also susceptible and serious epizodtics 
of coccidiosis due to E. stiede@ have been observed in cattle. . 


SPOROZOA 431 


Eimeria (Coccidium) Schubergi—This coccidium occurs in 
the intestine of a common myriapod (thousand-legged worm), 
Lithobius forficatus. It is the organism in which Schaudinn 
worked out the life-cycle now regarded as typical for Eimeriadz,. 
and which corresponds very closely to that of E. stiede. (See 
Fig. 80, page 162). 

Hemoproteus Columbe.—Celli and Sanfelice in 1891 ob- 
served this organism in the red blood cells of doves. It is widely 
distributed as a parasite of wild doves and has been found in 
Europe and in North and South America. -The life-history of the 
parasite in the vertebrate host and its mode of transmission by 
flies of the genus Lynchia has been most fully studied by Aragoa.} 
In the circulating blood of doves the organism is most commonly 
seen as a large crescent-shaped structure occupying most of the 
interior of an erythrocyte and crowding the nucleus of the latter 
to one side or encircling it. The outline of the erythrocyte and 
the outline of its nucleus are not distorted. The parasites are 
definitely recognizable as females and males, macrogametocytes 
with granular, deeply staining cytoplasm and microgametocytes 
with a paler cytoplasm. When these are ingested by the fly along 
‘ with its blood meal, the gametes arise, fertilization takes place 
and there is produced a creeping odkinete which apparently does 
not penetrate the intestinal wall in the fly or indeed undergo any 
further development there. It gains the blood stream of anew 
host, especially young nestlings, when the fly bites them. It is 
taken up by a leukocyte which comes to rest in the pulmonary 
~ capillaries of the young bird. Here the parasite produces a very 
large cyst and divides to form very numerous minute sporozoits. 
When the cyst bursts these sporozoits gain the blood stream, 
penetrate erythrocytes and grow to produce the gametocytes 
again. The asexual cycle of schizogony seems to be lacking. 

This organism is important as a typical example of Hemo- 
.. proteus, as it is the one species of this genus in which the life cycle 
has been most completely studied. 

! Archiv f. Protistenkunde, 1908, Bd. XII, 8. 154-167. 


432 SPECIFIC MICRO-ORGANISMS 


Fic.‘190.—Hemoproteus columbe. ta to 3a, Development. of the f 

_&- L , : emal - 
site in the}blood of the dove; 1b to 30, development of the male parasite a ihe ead 
of the dove; 4a, 4b, 5a, 5b, 6 to 12, development in the digestive tube of the fly 


(Lynchia); 13 to 20, development of the parasite inside leuk i 
the dove. (After Aragao.) WROTE Sethe lang-ak 


' SPOROZOA 433 


Hemoproteus (Halteridium) Danilewskyi—Grassi and 
Feletti! first clearly recognized this organism as a definite malarial 
parasite of birds. It is widely distributed and has been found i in 
very many different birds, including sparrows, doves, owls, robins, 
blackbirds and crows. The life history is incompletely known. 
In the blood of the infected bird the organism first appears as a 
small oval or lance-shaped body within the cytoplasm of an ery? 

throcyte. This enlarges, without distorting the outline or dis- 
placing the nucleus of the blood-cell, and stretches along one side 
of the cell. It curves about the nucleus and is enlarged at either 
end when fully developed. Two types, macrogametocytes and 


Fic. 191.—Hemoproteus danilewskyi. A and B, Fresh triple infection of red 
blood cells. C, D and E, Growing parasites, the last two showing vesicular nuclei. 
F, Full-grown halteridium with two nuclei. (After Doflein.) 


microgametocytes, are easily recognizable in stained prepara- 
tions. If blood containing these mature halteridia is diluted 
with citrated salt solution and studied under the microscope the 
‘further changes in the sexual cells may often be followed. Each 
gametocyte bursts the erythrocyte enclosing it and assumes a 
rounded outline. In the microgametocyte the protoplasmic 
granules exhibit violent agitation and several fine filamentous | 
~ processes suddenly shoot out from its periphery and lash about. 
After a few moments these microgametes separate completely 
and rapidly swim away. Meanwhile, the macrogametocyte has . 
escaped from its erythrocyte and come to rest in a rounded condi- 
tion. A microgamete approaches and penetrates the macro- 
gamete, and in a few minutes this fertilized sphere elongates into 
a curved spindle and actively creeps over the slide. It is then 


1 Centralbl. f. Bakt. 1891, Bd. IX, S. 403-409; 429-433; 460-467. 
28 


434 SPECIFIC MICRO-ORGANISMS 


known as the odkinete. Further development 
has not been observed, but there can be little 
doubt that the further stages of sporogony and 
also the unobserved stages of schizogony in the 
bird are somewhat. analogous to those of H. 
11 }\ columbe or to those of the plasmodia of human 
Wi) malaria. Whether the halteridia which occur in - 
various species of birds are all of one species cannot 
be decided without further investigations. 
Hemoproteus (Leukocytozoén) Ziemanni.— 
This organism was doubtless seen by Danilewsky 
in 1890.1 Ziemann in 1898 described it as a 
Fic. 192.— parasite in the blood of hawks. Its known life 
ee esis history is very incomplete, and even the nature 
ziemanni. Macro- of the blood cell containing it is somewhat” 
eo, Pe dgueeenl, . ie youngest stage observed in the 


microgametoc y.te 


DB otsen is vie ‘blood is a small oval parasite? situated at the side 
Schaudinn.) of the nucleus of the blood cell. The latter appears 


Fic. 193.—H e@moproteus (Leukocytozoén) ziemanni. A, Formation of[micro- 
gametes from the microgametocyte; B, Fertilization of the macrogamete by one of 
the microgametes swarming about it. (From Doflein after Schaudinn.) 


1 Centrabl. f. Bakt., 1891, Bd. IX, S. gor, Fig. 1. 
2 The description here given is derived in part from unpublished observations 


by Novy and MacNeal. See Proc. Soc. Exp. Biol. and Med., 1904-05, Vol. II, 
pp. 23-28; American Medicine, 1904, Vul. VIII, pp. 932-934. 


SPOROZOA | 435 


a 


to be an erythroblast, an immature red blood cell in which there is 
little or no hemoglobin. As the parasite enlarges, the host cell be- 
comes swollen and its nucleus much flattened and distorted. The 
parasite itself grows long and rather slender and is differentiated 


” 


Fic. 194.—Hemoproteus (Leukocytozoin) ziemanni in the blood of an ow! witha, 
pure infection. A, Young parasite in an erythroblast. B, Growing parasite dis- ~ 
.torting the nucleus of the host-cell. Cand D, Further stages of growth with marked 
distortion of the nucleus and of the outline of the host cell. £, Full-grown macro- 
gametocyte. F, Macrogametocyte and microgametocyte in the same field. G, 
Formation of microgametes from the microgametocyte. (After microphotographs of 
Prof. F. G. Novy.) g 


to form either the male or the female gametocyte, readily dis- 
tinguished by their-appearance in stained preparations. Mean- 
while, the host cell becomes very much elongated and pointed at 
the ends. The explanation of this peculiar distortion of the cell 


a 


442 SPECIFIC MICRO-ORGANISMS 


the salivary glands, from which they escape into the human host 
when the mosquito bites. The eyelet in Anopheles requires eight 
deve at a temperature of 28° to 30° C. At temperatures below 
17° C. the microgametes are not produced. » 
Development of the estivo-autumnal parasite through ‘hie 
stages of schizogony has been obtained by Bass and Johns! in the 
test-tube, in a medium consisting of defibrinated blood to which 
0.5 per cent~glucose has been added. They were able to keep the 
organisms alive for ten days at a temperature of 40° C., during © 


Fic. 205.—Section through salivary gland “of Anopheles showing numerous 
. sporozoits of Plasmodium falciparum. 1, Fat bodies; 2, gland duct; 3, sporozoits of 
Plasmodium; 4, Secretion in the gland cells. (From Dojiein after Grassi.) 
_ which period the developmental cycle was repeated four or five 
times. Their findings have been confirmed by other ‘investi- 
gators. More recently Joukoff? has reported partial development 
in the test-tube, of the cycle of sporogony in the case of Pl. falci- 
parum, and greater success with Pl. malaria. 

Plasmodium Vivax.—The parasite of tertian malaria is. dis- 
tinctly different from the estivo-autumnal parasite. The young 
merozoit is 1 to 24 in diameter and practically not to be distin- 


1Joun. Exp. Med., 1912, Vol. XVI, pp. 567-570. 
2 Compt. Rend. Soc. Biol., 1913, Vol. LX XIV, pp. 136-138. 


SPOROZOA 443 


guished, but very early in its growth it becomes actively ameboid 
and extends irregular and slender processes into the protoplasm 
of its host cell. As the parasite enlarges, the erythrocyte, often 
but not always, becomes swollen, paler, and shows a coarse granu- 


Fic. 206.—Plasmodium vivax. Stages of growth in the asexual cycle, commonly 


seen in the peripheral blood. Three of the cells show granules in the hemoglobin, 
the stippling of Schiifiner. X2200. (After Doflein.) 


lation, the stippling of Schueffner. The parasite often attains 
a diameter greater than that:of the average blood cell before it 
segments. The segmentation gives rise to from 15 to 30 mero- 
zoits which enter new erythrocytes and begin the cycle anew. 


Fic. 207.—Plasmodium vivax. Multinucleated Fic. 208.—Plasmodium 
stage preceding division and the stage of multiple vivax. Double infection of 
division (sporulation); found in the blood just be- a red blood cell which is 
fore and during a chill. X2200. (After Doflein.) considerably enlarged as a 

: result; Schiiffner’s stippling 
slight. X2200. (After 
Doflein.) 


This complete cycle of schizogony takes place in the peripheral 
‘circulation and requires almost exactly 48 hours. 

The young parasites destined to become gametocytes ex- 
hibit relatively less ameboid movement. Their pigment exists as 
large granules, some of them even rod-shaped. The macrogame- 
tocyte attains a diameter of 15 to 25u and usually destroys its 


444 SPECIFIC MICRO-ORGANISMS 


erythrocyte and escapes from it entirely. The cytoplasm stains 
deeply with methylene blue. The microgametocyte is smaller 
with paler cytoplasm. The development of the parasite in the 
mosquito (Anopheles) is wholly analogous to that of PI. falci- 
parum, although there are some slight morphological differences 
observed, Development ceases at temperatures below 16° C. 

Plasmodium Malarie.—The young quartan parasite is not 
‘characteristic, but in its growth it soon stretches ‘as a band across 
the erythrocyte. Later it almost fills the cell and then segments, 
producing 6 to 14, most often 8, merozoits. Tbe infected erythro- 


Fic. 209.—Plasmodium vivax. Stages in growth of the sexual cells (gameto- 
cytes). A and B, Young sexual cells distinguished from the agametes by the ab- 
sence of vacuoles and the more regular outline, C, Full-grown macrogametocyte. 
D, Full-grown microgametocyte. 2200. (After Doflein.) 


cyte is not enlarged or distorted nor does it become pale or show 
granulation. The gametocytes, when stained, are not very 
different in appearance from the asexual cells. In the living 
preparation they show much more active protoplasmic move- 
ment. The sexual cycle takes place in Anopheles and agrees 


very well with that of the other two malarial parasites, as S far as 
it has been studied. 


Malaria is probably the most important as well as the most 
well-known human disease due to protozoa. It is characterized 
by recurrent paroxysms of fever with afebrile intervals, progress- 
ive anemia and weakness, with the accumulation of a dark brown 
or black pigment in the spleen and liver. This pigment is pro- 
duced by the parasites and set free into the blood when they 


i 
a 

Ree 
Pe 


( 


SPOROZOA ~ 23 445 


.segment. The estivo-autumnal malaria caused by PI. falci- 


parum shows a somewhat irregular and not very characteristic 
fever curve, but usually there is fever every day (quotidian fe- 


-ver). The tertian fever due to infection with Pl. vivax is char- 


Fic. 210.—Plasmodium malarie. Stages of the asexual cycle in the circulating 
blood. Note the absence of granulation from the hemoglobin and the uniform size 
of the red blood cells. 2200. (After Doflein.) , 


acterized by febrile attacks recurring at intervals of 48 hours and | 
bearing a very definite relation to the asexual cycle of the para- 
site. The segmentation of the plasmodium is coincident with 


Fic. 211.—Plasmodium malarie. Sexual cells in the circulating blood. A, 
Young gametocyte. B, Full-grown macrogametocyte. C, Full-grown microgame- 
tocyte. 2200. (After Doflein.) 

4 


the chill and the rise in the patient’s temperature. In quartan 
malaria due to infection with Pl. malarie, the fever recurs at 
intervals of 72 hours, again at the stage of segmentation in the 
asexual cycle of the parasite. Obviously an association of two 


‘ 


4 36 SPECIFIC MICRO-ORGANISMS 


is unknown, but it may be due to the mechanical streaming of the 
blood acting upon the bladder-like cell which has been deprived of 
elasticity by the destructive action of the parasite. The further 
stages in the cycle of sporogony are unknown. An asexual 
multiplication probably occurs in some internal organs of the bird. 
Fantham has observed schizogony in the spleen of Lagopus 
scoticus, the red-game grouse of Scotland, infected with a similar 
parasite Leukocytozoon tovait. 


sage 

Fic. 195.—Diagram of the developmental cycle of Proteosoma. 1. Sporozoit 
entering an erythrocyte; I, 2, 3 and 4, the cycle of schizogony; 5, macrogameto- 
cyte; 54, microgametocyte; 6, macrogamete; 6a, formation of microgametes; 7- 
fertilization; 8, odkinete; 9, formation of sporoblasts (in mosquito); 10, forma- 
tion of sporozoits; I1, sporozoit. (From Doflein after Schaudinn.) 

Proteosoma (Plasmodium) Preecox.—Grassi and Feletti de- 
scribed this malarial parasite of birds’ and designated it as Hem- 
ameba precox.1 The parasite is very common in the blood of 
small birds, such as sparrows, robins and larks, in all parts of the 
world. The cycle of schizogony is completed in the peripheral 
, circulation. The small merozoit or agamete enters an erythro- 


1 Centrabl. f. Bakt., 1891, Bd. IX, S. 407. 


SPOROZOA (437 


cyte and enlarges, retaining its oval or circular form. The nucleus 
of the host cell is pushed out of position but its form is not ma- 
terially altered. The full-grown parasite segments, producing: 
10 to 30 merozoits and leaving behind a small residual body con- 
taining the accumulated pigment, thus completing the asexual 


Fic. 196.—Proteosoma precox in the blood of a field lark (Glauda arvensis). 
A, Young parasite in a blood cell., B, Half-grown parasite which has pushed aside 
the nucleus of the erythrocyte. C, Parasite with clump of pigment and many nuclei. 
The nucleus of. the erythrocyte has been lost (uncommon). D, Division into eight- 
een merozoits. (From Doflein after Wasielewski.) yo 


cycle, which may be repeated many times. After a time some 
of the growing parasites become differentiated to form macro- 
gametocytes and microgametocytes, which are kidney-shaped and 
do not divide nor undergo further development in the vertebrate 
host. When the blood is drawn and diluted with citrated salt so- 


Fic. 197.—Midgut of a culex mosquito, covered with odcyts of Proteosoma precox. 
V, Vasa malpighii. (From Doflein after Ross.) 
% 


lution, or taken in by a mosquito, four to eight microgametes are 
formed just as has been described for H. columbe. They are very 
slender actively motile spindles without flagella. Fertilization 
of the macrogamete and the production of an odkinete takes 
place in the usual manner. The latter penetrates the intestinal 
epithelium of the mosquito (Culex sp.) and enlarges to produce 


x‘ 


438 SPECIFIC MICRO-ORGANISMS 


a spherical cyst filled with an enormous number of thread-like 
sporozoits. These escape into the body cavity of the mosquito 
as the cyst bursts, and are generally distributed throughout the 
‘body of the insect. They assemble, probably as a result of some 
_chemical stimulus, in the salivary glands of the mosquito, whence 
they are injected into the wound as the insect bites, and at once 
invade erythrocytes to begin the cycle of schizogony. 

' The discovery of the sexual cycle of pro- 
teosoma in the mosquito and the conclusive 
proof that this form of bird malaria is trans- 
mitted by a mosquito stands to the ever- 
lasting credit of Ronald Ross. His brilliant 
discovery made in India in 1898, pointed 
the way to the solution of the whole problem 
‘of the transmission of the malarial diseases 
and their practical restriction. 

Proteosoma is a favorable parasite for 
class study, as it is readily transmitted from 
bird to bird (sparrows or canaries) by in- 
jection of infected blood, and the parasites, 
often become very numerous ‘in the blood. 
Fic. 198.—Oécyst of ‘There seems to be no good reason for placing 


Proteosoma precox, de- a A z 
veloped on the intestine this organism in a separate genus from the 


of Aedes (Stegomyia) co- human malarial parasites. 
opus, showing numerous : 

sporozoits. (From Do- Plasmodium Falciparum (Przcox).— 
fein after Neumann.) T averan in 1880 discovered the first ma- 
larial parasite in the blood of man and correctly interpreted 
bis observations. The distinctions between the three species 
was recognized by Golgi, and the life history of the parasites 
and especially their relation to mosquitoes and insects in gen- 
eral has been most thoroughly studied by Grassi! Pl. falcip- 
arum is the parasite of estivo-autumnal or pernicious malaria 
of man. The young organism is 1 to 1.54 in diameter. It pene- 
trates a red blood cell and enlarges. A vacuole appears in the 


1 Grassi: Die Malaria, IIte Auflage, Jena, 1901. 


SPOROZOA 439 


center, giving the parasite the appearance of a signet ring, the 
setting being. represented by the nucleus or chromatin granule 


A B Cc 


Fic. 199.—Plasmodium falciparum, forms in the asexual cycle (schizogony). 
A, Multiple infection of an erythrocyte, showing signet rings and parasites attached 
to the external surface. Band C, Growing parasites with Mauer's granules in the 
erythrocytes. D, Growing parasite without granulation of the hemoglobin. E, 
Half-grown parasite showing pigment. F, and G, Multiple division (sporulation), 
rarely seen in the peripheral blood. (After Doflein.) ; 


which stains violet red with the Romanowsky stains. The 
parasite attains a diameter of about 6y, when it segments to 
produce 7 to 16 merozoits or 
agametes which enter new ery- 
throcytes and repeat the cycle. 
The larger stages of this cycle of 
schizogony are rarely seen in the 
peripheral circulation, and the 
segmentation of the parasite oc- _ ' 
curs in the capillaries of the in- ee eee ee 

i ’ g-numerous di 


ternal organs. The cycle prob- viding forms of the non-pigmented type 


<n . of Pl.> falciparum. (Stained prepara- 
ably - requires 48 hours for its tion.) (From Doflein after Mannaberg.)~ 


completion. The erythrocyte 
is not enlarged by the growth of the parasite within it but tends 
tather to become smaller. Maurer has observed an irregular 


440 ; SPECIFIC MICRO-ORGANISMS 


granulation of the erythrocytes. Why the cells containing the 
‘larger forms should remain in the internal capillaries of the body 
is not definitely known. 


x 


Fic. 201.—Plasmodium falciparum. Stages in the development of he gametocytes | 
(crescents). 2200. (After Doflein.) 


The gametocytes develop by the growth of ordinary merozoits, 
which become erescentic early in their development and differen- 


A B 


Fic. 202.—Sections through the stomach wall of Anopheles showing stages in 
the development of Pl. falciparum. A, Fixed a few hours after the infective feeding, 
showing odkinetes within the lumen and two in the cuticula of the epithelium. B, 
Fixed a few days after the infective feeding, showing the partly grown odcyst in the 
stomach wall. F, Fat surrounding the stomach; em, tunica elastico-muscularis; e, 
epithelium; c, cuticula; J, lumen of stomach. (From Doflein after Grassi). 


tiated into deeply staining macrogametocytes and pale-staining 
microgametocytes. These are produced especially in the bone 
marrow and they circulate in the peripheral blood. Further 


SPOROZOA 441 


. 


development takes place when the blood is taken into the stomach 
of a mosquito of the genus Anopheles. Here the microgametes, 
slender actively motile threads, are given off by the microgam- 
etocyte and fertilize the macrogametes, producing odkinetes, 


Fic. 203.—Digestive tract 
of Anopheles, the stomach of 
which is covered with numer- 
ous odcysts of Pl. falciparum, 
viewed from the left side. c, 
Cloaca; s, stomach; o, odcysts 
of Plasmodium; mt, malpighian 


tubules; sb, sucking bladders; 


sg, salivary gland. (From Do- 
flein, modified after .Ross and 
Grassi.) _* 


which actively penetrate the epithelium. 

In the wall of the mosquito’s stomach 
each odkinete gives rise to a rapidly 
growing cyst and within this an enor- 
mous number of very slender sporo- 
zoits are developed. The ripe’ cyst 

bursts into the body cavity and the 
sporozoits become generally distributed 
throughout the hody of the insect and: 
later assemble in the secreting cells of 


Fic. 204.—Plasmodium falciparum. 
Ripe sporozoits arranged about residual 
bodies within the odcyst, cut in various 
directions (7 to 8 days after infection of 
the mosquito). (From Doflein after 
Grassi.) : 


446 SPECIFIC MICRO-ORGANISMS. 


or more crops of parasites reaching maturity at different times 
may give rise to a variety of fever curves. \ 

The diagnosis of malaria is most conclusively established by 
recognizing the parasites in the blood of the patient. One 
should examine a fresh drop of blood, unstained, under the 
microscope, and also thin films of blood stained with some 
one of the Romanowsky stains. The parasites may be very 
scarce in old cases and especially in those patients who have been 
treated. 

The mosquitoes which transmit human malaria were first 
recognized by Ross and have been most thoroughly studied by 
Grassi. The mosquito:is capable of causing malaria only after 
it has fed upon a person harboring the pardsite in his blood. 
The members of the genus Culex, the most common mosquitoes, 
do not permit the development of the plasmodia within them, but 
this occurs, so far as is known, only in certain species of the genus 
Anopheles. A. maculipennis in Europe and A. quadrimaculatus 
in America appear to be the most important species. They are 
easily recognized by the four small black spots on each wing due 
to a relative accumulation of pigmented scales in these situations. 
The members of the genus Anopheles are readily distinguishable 
from Culex by the form and arrangement of their eggs. the form 
and position of the larve and by the general form and structure 
of the adult insect, as well as its posture when at rest. 

The restriction and prevention of malaria is founded upon the 
knowledge of its nature and its mode of spread. The measures 
include (1) the destruction of malarial parasites in man by thor- 
ough treatment of the disease with quinine, (2) destruction of 
mosquitoes and mosquito larve and the drainage, oiling or screen- 
ing of their breeding places, and (3) exclusion of mosquitoes 
from contact with infected persons and also from contact with 
healthy persons, by the use of screens. The thorough application 
of these measures has demonstrated the possibility of effectively 
controlling this disease even in the tropics. 

1 Fermi and Lumbau: Centrbl. f. Bakt., 1912, Bd. LXV, pp. 105-112. 


447 


SPOROZOA 


(note position), 
(From Jordan 


¥ 
vee 


u 
a a 
oO y 
a 24 & 
S tw a 
, —_. 
< Yr 


amr SINS \ 


ee) 
SS 


wings, heads showing antenne and palpi. 


Culex 


Fic. 212.—Comparison of Culex and Anopheles. 


position of insects at rest, 
after Kolle and H etsch.) 


448 . SPECIFIC MICRO-ORGANISMS 


Plasmodium Kochi—This is a malarial parasite which causes 
a mild fever in monkeys. It is not transmissible to man. Other 
species of malarial parasites have been recognized in these animals. 

Babesia! Bigemina.—Smith and Kilborne . discovered this 
organism in the red blood-corpuscles of cattle suffering from 
Texas fever. The parasite is pear-shaped, 2 to 4u long and 1.5 to 
2u wide and usually occurs in pairs within the erythrocytes. The 
cytoplasm is quite clear without granules or pigment and contains 
one or two chromatin bodies. Minute ameboid forms are ‘also 
found. Multiplication apparently takes place by longitudinal] 


Fic. 213.—Babesia bigemina. Characteristic forms in the peripheral blood of cattle. 
X2000. (After Doflein.) 


division of the pear-shaped forms as well as by multiple division 
of the ameboid forms. Macrogametocytes and microgametocytes 
have been recognized. The transmission of the parasite from 
animal to animal is effected by the cattle tick, Bodphilus bovis, 
(Rhipicephalus annulatus) as was conclusively demonstrated by 
Smith and Kilborne, the first instance in which such a relation 
was proved for any blood-sucking invertebrate. The details of 
the life cycle in the tick are unknown. It is certain however 
that the infection is conveyed to the next generation of ticks 


1 The generic name Pyrosoma bestowed by Smith and Kilborne in 1893 is incor- 
rect, because this is the name of a genus of marine animals belonging to the Tuni- 
cata. Babesia proposed by Starcovici in 1893 has the next claim to priority. 


SPOROZOA 449 


through the eggs and that these young ticks are capable of in- 
fecting cattle. Renewed investigation of the parasite is much to 
be desired... 

Texas fever is a very important disease of cattle in the southern 
United States and a similar disease occurs in Europe, Africa and 
South America. Young cattle usually survive the disease and 
become immune. Older cattle imported into the endemic area 
contract Texas fever and usually die of it. Immunity may be 
conferred by injecting blood which contains a small number of 
parasites, taken from an animal which has passed the acute stage 
of the disease. Restriction of the Texas-fever area in the United 
States is slowly progressing as a result of systematic eradication 
of the tick. 


Babesia Canis.—This organism occurs in the blood of dogs: 


suffering from the ‘so-called malignant jaundice, and has been 
carefully studied by modern methods by Nuttall and Graham- 
Smith and later by Breinl! and Hindle. In morphology and life 
history it agrees with B. bigemina as far as these have been worked 
out, but B. canis is incapable of infecting cattle. The infection 
is transmitted to dogs by several different species of ticks. 

Gregarina Blattarum.—This organism lives as a parasite in 
the intestine of the common cockroach Periplaneta orientalis, and 
is therefore liable to be found in human food, and at times in 
specimens from human cases submitted to microscopic study, 
probably because of accidental presence of cockroaches in the 
containers employed. The vegetative cells are elongated, often 
attached together. The spore ‘cyst results from the union of two 
cells and the subsequent repeated division of the fertilized cell 
to produce an enormous number of spores. These spores are 
discharged from the cyst when it enters a fluid medium: When 
fully developed, each spore contains eight sporozoits. 

Nosema Bombycis—This organism was discovered by Naegeli 
in 1857. It is an example of the Neosporidia and is of peculiar 
interest as the cause of pébrine, the disease of silkworms studied 


1 Ann. Trop. Med. and Parasitol., Vol. II, pp. 233-248. 
29 


450 SPECIFIC MICRO-ORGANISMS 


by Pasteur in 1866-1870, and largely eradicated by application 
of the methods devised by him as a result of his investigations. 
The spore of NV. bombycis is 1.5 to 24 wide by 3 long. If treated 
with nitric acid it swells and reaches a length of 6m and extends 
a slender thread which may be top long. The spore is ingested 


Fic. 214.—Gregarina blattarum. I, Two individuals stuck together. II, Cysts 
with conjugated cells and developing spores. IIIA, Unripe spore with undivided 
contents. IIIB, Ripe spore with eight sporozoits; ek, ectoplasm; en, endoplasm; 
cu, cuticula; pm, protomerit; dm, deuteromerit; , nucleus; 7”, spores; rk, residual 
body; sk, sporozoits. (From Doflein after R. Hertwig.) 


by the silkworm and in its intestine the ameboid parasite escapes 
and penetrates the epithelium. It may pass to any part of 
the host to undergo its further development. Multiplication 
of the small.rounded agamete results in the formation of long 
chains of oval bodies inside a cell of the host. From these the 


SPOROZOA 451 


spores are again produced. Pébrine is a disease of the greatest 
importance to the silkworm industry. It is effectively restricted 


Fic. 215.—Nosema bombycis. ‘Section of intestinal epithelium of silkworm | 
showing spores of Nosema and also the peculiar multiplication resembling the 
growth of a mold. (From Doflein after Stempell.) (See also Fig. 83, p. 165.) 


by a careful microscopic examination of all the silkworm eggs 
and the exclusion and destruction of all those in which the para- 
site exists (Pasteur’s method). 


CHAPTER XXX 


CILIOPHORA 


Paramecium Caudatum.—This is the most common infusor- 


Fic. 216.— Paramecium 
caudatum. K, Macronucleus; 
NK, micronucleus; C, gullet; 
N, food vacuoles; CV, contrac- 
tile vacuoles. (After Doflein.) 


ian met with in stagnant water. Its 
length varies from 120 to 3254. The 
cell is spindle-shaped with a deep oral 
groove which takes a spiral course on 

one side of the body. The surface is 
thickly set with active cilia. Food 
particles are swept into the oral 
‘groove, enter the cytoplasm at its 
bottom and circulate in the cell within. 
food vacuoles. Near the center of the 
cell is a large macronucleus and near it 
asmaller micronucleus. Multiplication 
takes place by simple longitudinal or 
oblique division. 

Conjugation is isogamic. The sim- 
ilar conjugating cells adhere to each 
other, the micronuclei divide twice and 
three of the four nuclei thus produced 
disintegrate, as does also the macro- 
nucleus. The remaining micronucleus 
divides into two and one of these 
passes into the other conjugating cell 
in exchange for a simliar element. 
The newly acquired element unites 
with the element which remained be- ~ 
hind to form the new nucleus. The 
new nucleus divides three times in suc- 

452 


living cells. For example Jen- 


CILIOPHORA 453 


cession to form eight nuclei, of which four enlarge to become 
macronuclei, one remains as a micronucleus and three disin- 
tegrate and disappear. The one micronucleus then divides by 
mitosis and the cell divides to form two paramacia, each contain- 
ing one micronucleus and two 
macronuclei. The next division 
gives rise to cells containing the 
normal number of nuclei, one 
micronucleus and one macro- 
nucleus. 

The paramecia are lagre sapro- 
phytic organisms, easily kept under 
cultivation in the laboratory, and 
they have been very extensively 
studied. Many conceptions 
founded upon these studies are 
considered to have a broad bear- 
ing upon the physiology of all 


. 1 _ 
aMge has found that Cone Fic. 217.—Paramecia drawn at 


gation serves two ‘purposes, (1) to the same magnification. A, Para- 
mecium caudatum. B. Paramecium 


“provide chemical stimulation of putrinum. (From Doflein afler Sche- 


cell division and (2) to insure va- kof). 
riety in the descendants. The variety in the descendants is a 
result of the exchange of nuclear material. Calkins? has dis- 
covered a specialization of function in paramecium in respect 
to conjugation and concludes that in some of the descendants 
of an ex-conjugant the ability to conjugate is in abeyance, 
thus suggesting a resemblance to the somatic cells of a metazo6n, 
while other descendants retain this function and are therefore 
analogous to the germ cells of a metazoon. 

Three other species of paramecium are recognized, namely, 
P. aurelia. P. bursaria and P. putrinum. 


1 Harvey Lectures, 1911-12, pp. 256-276. 
* Proc. Bre Exp. Biol. and Med., 1913, Vol. X,,pp. seb, 


i 


454 SPECIFIC MICRO-ORGANISMS 


Opalina Ranarum.—This is a common, parasite in the in- 
testine (cloaca) of the frog. It reaches a large size, 600 to 800y 
in diameter, is flattened and somewhat irregular in outline. The 
ectoplasm is striated and there are very many nuclei in the in- 
terior of the cell. In the springtime, as the frogs enter water to 
spawn, the parasites divide rapidly and give rise to cysts 20 to 4ou 
in diameter. These escape into the slime and are ingested by 
_ the growing tadpoles. In the cloaca the cells escape from the 
cysts. They are differentiated into male and female gametes and 


Fic. 218.—Opalina ranarum, showing the numerous vesicular nuclei. A, Ordinary 
form. B, Dividing form. (From Doflein after Zeller.) 


fuse to form one cell which grows and multiplies in the developing 
frog. 
Balantidium Coli—tThis parasite of the human intestine was 
described by Malmsten in 1857. Its normal habitat seems to 
be in the large intestine of swine, where it is commonly found in 
large numbers. The cell is a short oval, 50 to 7on wide and 
7° to 100m long, rarely larger. Its surface is covered with active 
cilia, and there is a short oral groove at the anterior end. The 
cytoplasm contains drops of fat and food vacuoles, often red 
blood cells and leukocytes of the host. The principal nucleus 


CILIOPHORA 455 


is kidney-sShaped and the accessory nucleus lies in contact with 
it. Multiplicaton takes place by simple transverse fission. 
Conjugation and cyst formation have been observed. 


Fic. 219.—Balantidium coli. A, Fully developed individual, showing the 
nucleus above at the right and a food particle below. BandC, Division stages. D, 
Conjugation. (From Doflein after Leuckart.) 


Bal. coli is sometimes found in man in cases of intestinal dis- 
order with diarrhea. Its possible causal relation to the patho- 


Fic. ed6-—Section through the intestinal wall in a case of enteritis due to 
Balantidium. S,Serosa; M. Muscularis; B, Balantidia. (From Doflein after Solow- 
jew.) 


logical condition is not conclusively ascertained. In some in- 
stances the cells of Balanlidium have been found deeply situ- 


456. 


ated in inflanned intestinal wall. 


Fic. 221.—Sphero- 
phrya pusilla within a 
paramecium. At one 
place there are four 
parasites and a fifth is 


escaping. 
one of the parasites is 
just penetrating the 
host, and a single para- 
site is seen near the 
center of the parame- 
cium. 
after Biitschli.) 


Higher up, 


(From Do flein. 


SPECIFIC MICRO-ORGANISMS 


Brooks! observed Bal. colt in 
several cases of dysentery in Orangoutangs 
in the New York Zoological Park and 
Brumpt? has been able to transfer balan- 
tidium infection from monkey to swine 
and back to monkey. Still there is perhaps 
some question as to the identity of the para- 


- sites found in man and in hogs. 


Balantidium Minutum.—Schaudinn in 

1899 observed this organism in the human 
feces. It is smaller then Bal. coli, the 
greatest measurements being 20 X 30m, and 
the oral groove extends more than half 
way. ‘back along the side of the cell. It 
probably occurs rarely in the human small 
intestine. Other species of balantidium have 
been described. 
_ Spherophrya Pusilla.—-This organism is 
of peculiar interest because it lives as a 
parasite within another protozoén, the para- 
mecium. The cell of Sph. pusilla is spheri- 
cal, 20 to 40m in.diameter, and provided with 
sucking tentacles and cilia when outside the 
body of the host. 

1 Proc. N. Y. Path. Soc., 1903, Vol. Ul, pp. 28-39. 

2 Compt. Rend. Soc. Biol., 1909, Vol. LX VII, pp. 103- 


105. 
u 


INDEX OF NAMES 


Abbé, 16 

Abbott, 110 
Adil-Bey, 388, 
Agramonte, 376 
Amoss, 393 
Anderson, F., 341 
Anderson, J. F., 294, 394 
Andrade, 348 
Appert, 6 
Aragao, 431, 432 
Aristotle, 3 
Arkwright, 396 
Arloing, 287 
Armato (d’Armato), 15 
Arnold, 67 
Arrhenius, 211 
Arustamoff, 257 
Ashburn, 393 
Ashford, 247 
Atkins, 180 
‘Atkinson, 306 
Audouin, 10, 243 
Austen, 404 
Avery, 270 
Axenfeld, 309 


Bacmeister, 378 
Bacon, 15 
Baehr, 304 
Baeslack, 392 
Baetjer, 377 
Bail, 212, 231 
Bang, 356 
Banzhaf, 215, 306 
Barbagallo, 160 
Bashford, 396 
Bass, 442 
Bassis, 10, 243 


Bastian, 4 
Bataillon, 313 


Bateman, 407 1 

Baumgarten, 144 

Becker, 285 

Behring (von Behring), 12, 215, 234, 292, 
293, 305, 307 

Berg, 245 


Besredka, 232, 343 4 


Beurmann (de Beurmann), 252 
Beyerinck, 14 e 
Biggs, 303 
Billroth, rz 
Blake, 269, 275, 311- 
Blanchard, 404 
Boidin, 285 
Bollinger, 254, 287 
Bolton, 94, 392 
Bordet, 211, 222, 223, 224, 235, 309 
Bradford, 396 
Brefeld, 238 
Breinl, 449 
Brem, 328 
Bretonneau, 302 
Brieger, 210 
Briscoe, 325 
Broadhurst, 167 
Brooks, 456 ~ 
Brown, J. H., 275 
Brown, L., 324 
_ Bruce, 334, 377, 497 
Brueckner, 297 
Brumpt, 456 
Buchanan, 167 
Buchner, 129, 219, 222, 225, 234 
Buetschli, 456 
Bumm,; 258 
Burdick, 316 
457 


s 


458 INDEX OF NAMES 


Burke, G. S., 295, 296 Dick, 257 
Burvill-Holmes, 328 Dickson, 296 
Buschke, 252 Dobell, 368 
Busse, 252 Doerr, 393 


Doflein, 157, 163, 167, 397, 404, 406, 408, 
416, 420, 426, 433, 439, 440, 
443, 444, 445, 448, 452 


Cagnaird-Latour, 6 
Calkins, 425, 453 


Calmette, 323, 328 
Carle, 290 

Carroll, 376 
Casagrandi, 160, 395 
Castellani, 407 
Cecil, 311 

Celli, 431 

Chace, 106 

Chagas, 411, 413 
Chambers, 403, 406 
Chatterjee 159, 415 
Chauveau, 234 


Donné, 10 
Donovan, 158 
Dorset, 97, 315, 392 
Douglas, 224 
Dreyer, 348 
Dubard, 313 
Duclaux, 14 
Ducrey, 312 
Dunham, 94 
Durham, 218 
Dusch, 6 | 

Dutton, 368, 369, 407 


Chester, 180 
Chevalier, 15 
Citron, 217, 222, 231, 236 


_Eberth, 343 

Ehrenberg, 4, 168, 337, 368 
Clark, H. W., 189, 195 Ehrlich, 211, 212, 214, 215, 216, 217, 
Clark, W. M., 89, 90 218, 220, 225, 234, 235, 236, 
Cohn, 210 302, 307 


Cohn, F., 5 Ehrmann, 380 

Cole, 268 Eichorn, 356 

Conn, H. J., 90, 180 Einhorn, 106 

Conor, 394 Elmassian, 407 

Conseil, 394 Emmerich, 364 

Cornet, 37, 38 Endo, 347 

Cornevin, 287 Erb, 261 

Councilman, 95, 262 Ermengem (van Ermengem), 53, 294, 
Couvy, 376 : 295, 362, 

Cox 253 Escherich, 337, 339, 340, 358 
Craig, 393 Esmarch, 114, 193, 360 
Cumming, 390 Evans, 406 

Curtis, 317, 340 Eyre, 335 
Danilewsky, 410, 434 Fabyan, 356 


d’Armato, 15 
Davaine, 10, 281, 419 
DeBeurmann, 252 
Delafield, 61 

Denys, 319 

De Schweinitz, 392 


Fantham, 409, 436 
Fehleisen, 271 
Feletti, 433, 436 
Fennel, 349 
Fermi, 446 
Ferran, 229, 366 


INDEX OF NAMES 459 


Feser, 287 

Finkler, 367 

Fischer, 155 

Fitzgerald, 353 

Flexner, 225, 263, 264, 352, 303 
Fhigge, 195 : 
Fontana, 379 

Forde, 407 

Forscher, 290 

Fortineau, 285 
’ Fracastorius, 9, 208 

Frankel, A., 266, 282, 293 
Freer, 330 

Friedlander, 266, 268, 340 
Frisch (von Frisch), 340 
Frosch, 388 

Frost, 180 

Fuller, 86, 186, 188 

Futaki, 372 


Gaffky, 343 

Gage, 189 

Galen, 8 

Galileo, 15 
Gamaleia, 367 
Garbat, 217, 236 
Garré, 277 

Gartner, 341 
Gatewood, 312 
Gemy, 256 

Gengou, 247, 224, 309 
Geppert, 75 

Gessard, 359 
Gibson, 306 

Gibson, 348 

Giemsa, 43. ~ 
Gilchrist, 242, 253, 357 
Goldberger, 394 
Golgi, 438 

Goodsir, 278 
Gordon, 16, 263, 335 
Gorham, 180 
Gorsline, 139 
Gougerot, 250, 251 
Graham, 297 | 
Graham-Smith, 449 


Gram, 45, 59 

Grassi, 159, 418, 433, 436, 438, 440, 44r, 
446 

Grawitz, 246 

Grondahl, 255 

Gruber, 218 

Grund, 366 

Guarnieri, 395 


Haffkine, 229, 334, 366 
Hamburger, 323 


. Hamerton, 407 


Hannum, 311 
Hansen, 326 
Harbitz, 255 
Harding, 180 
Harrison, 381 
Hartmann, 417, 421, 422, 423, 424, 425, 
430 
Harvey, 3 
Hauser, 358 
Heidenhain, 54 
Heim, 171, 338, 344 
Hektoen, 224, 252, 394 
Hellriegel, 14 
Hemenway, 322 
Henderson, 311 
Henle, 202 
Herbst, 312 
Herodotus, 7 
Hertwig, 450 
Hess, 106, 346 
Hesse, 1 
Hetsch, 447 
Hill, 35 


, Hindle, 371, 449 


Hippocrates, 8, 290 
Hiss, 52, 265, 266, 353 
Hoffman, 377, 392 
Hoffmann, 309 ~ 
Hoffman, E , 377, 378 
Hégyes, 230 

Hoki, 373 | 

Holmes, 274 


Holt, 322 


Homer, 8 


460 INDEX OF NAMES 


+ - 


Hornor, 311 Koch, 1, 11,12, 66, 96, 110, 116, 123, 124, 
Huebner, 262 202, 203, 266, 274, 281, 286, 
Hueppe, 96 399, 313, 318, 319, 343, 360, 362 
Huntoon, 52, 31m Kolle, 228, 229, 230, 283, 202, 334, 366, 
Hutchings, 195 388, 447 
Hyde, 252 Koplik, 332 

Kossel, 314 
Ido, 373, 376 ‘ Kraus, 216 
Inada, 373 Krauss, 40 
Inman, 348 _ Krumwiede, 167, 325, 347, 306 
Irons, 261 Kruse, 352, 
Israel, 254, 255 Kyes, 270 


Ito, 373, 376 
Laennec, 319 


Jaeger, 262 ‘ Lafar, 192 
Jeffer, 191, 192° Landouzy, 319 
Jenner, Edward, 12, 230, 395 Langenbeck (Von Langenbéck), 245 
Jenner, Louis, 42 Latzer, 133 ‘ 
Jennings, 453 Laveran, 12, 413, 417, 438 
Jensen, 337 : Lazear, 376 
Jochmann, 264 ! Leeuwenhoek, 3, 5, 9, 15 ; 
Johns, 442 Leishman, 42, 350, 371 
Johnson, 186 Lentz, 390. y 
Jones, 264 Leuckart, 455 
Jordan, 187 Levaditi, 377, 379, 380 
Joukoff, 442 : Lewis, G. W., 400 

: - Lewis, Paul A., 393 
Kahn, 328 Lewis, T. R., 400 
Kaneko, 373 : Liborius, 128 
Kanthack, 256 Lichtheim, 237, 238 
Kellerman, 189 Liebig, 7 
Kerr, J., 133, 288, 356 Lindemann, 429 
Kessler, 328 Lingelsheim (Von Lingelsheim), 273, 
Kilborne, 448 ‘ 276, 292 
King, 392 ? Lipman, 183 
Kinghorn, 410 Lister, 11 
Kircher, 9 Lloyd, 410 
Kirkbride, 39. Loffler, 1, 42, 52, 96, 298, 322, 342, 354, 
‘Kitasato, 12, 215, 290, 292; 330, 332 388 \ 
Kitt, 287 Longley, 188 
Klebs, 11, 271, 275, 298 Léschj 12, 160, 421, 423 ’ 
Klegg, 256, 419. Lowenstein, 319 
Kligler, 180, 353 Lubs, 89, 90 
Klimenko, 310 Luer, 99 
Knapp, 369, 370 Luetscher, 340 


Kneass, 280 Lumbau, 446 


INDEX OF NAMES 461 


McBryde, 392 Moses, 7 
McClintock, 75 Mubhlens, 377 
McCrae, 139 Miller, 4 
McFadyean, 205 é Muns, 270 
McFarland, 344 Murphy, 396 
McIntosh, 377 Musgrave, 256, 419 


MacNeal, 44, 106, 121, 122, 133, 200, 


242, 311, 325, 356, 377, 382, Niageli, 449 
397, 398, 401, 402, 403, 405, Needham, 5 


499, 410, 434 Negri, 389 
MacNee, 376, 377 Neisser, 222, 258, 276, 290, 326 
McNeill, 261 Neufeld, 224 | 
McWilliams, 347 Neumann, 438 
Mackie, 407 Nicolaier, 290 
Madsen, 211 Nicolle, 388, 394, 416 
Mafucci, 313 Nocard, 13, 342, 388 
Major, 271 Nocht, 42 
Mallory, 95, 262, 311 Noguchi, 13, ror, 264, 369, 373, 374; 375; 
Malmsten, 454 376, 377; 387, 393 
Manneberg, 439 Novy, 13, 37, 99, IOI, 122, 130, 131, 
Marchoux, 371 I5I, 152, 155, 158, 339, 341, 
Marshall, 174 369, 370, 397, 398, 402, 403, 
Marzoli, 15 406, 409 410, 434, 435 
Massee, 241 Nuttall, 13, 210, 234, 287, 360, 379, 371; 
Matschinsky, 310 : 449 , ; 
Maurer, 439 Nuzum, 304 
Mayer, 56 : 
Mesnil, 413, 417 Obermeier, 11, 13, 368 
Metchnikoff, 214, 219, 224, 232, 234, Ocrgel, 364 

343 Ogston, 271, 275 
Metzner, 429 Olitsky, 394 
Michel, 247 Ophiils, 242 
Migula, 5, 148, 150, 152, 153, 180, 316, Opie, 377 

337 Orth, 61 
Milne, 368 Osumi, 372 
Miquel, 75 : 

. Mitchell, 270 Pakes, 192 

Moffitt, 242 Pandy, 264 :: 
Mohler, 356 Pappenheimer, 349, 377 
Miller, 47, 51, 327 Park, 300, 301, 303, 306, 311, 325, 354 
Montague, 229 ‘ Passini, 129, 209, 230, 234 
Montgomery, 252 Pasteur, 1, 3, 4, 6, 7, II, 33, 129, 209, 
Moore, 186, 189 230, 234, 266, 275, 284, 286, 
Morax, 309 329, 391, 459, 451 
Moritz, 329 Peacock, 377 


Moro, 323 Peppard, 253 


462 INDEX OF NAMES 


Perkins, C. F., 252 Ross, R-, 368, 437, 438, 441, 446 

Perkins, W. A., 403, 406 Rothberg, 353 

Petri, 112 Rouget, 406 

Petruschy, 254 Rous, 396 i 

Pettenkofer, 8, 364 Roux, 123, 301, 306, 388 

Pfeiffer, 101, 219, 220, 234, 282, 293,311, Ruediger, 252 | 

363, 364 Rufus of Ephesus, 332 

Pirquet, 226, 233, 261, 323 Russell, 347, 348, 350 

Plaut, 239, 245, 246, 247, 248, 240 _ Rymowitsch, 310 

Plenciz, 9 

Plotz, 394 Sabouraud, 250 

Pollender, 10, 281 Sacharoff, 371 

Poor, 389 Sailer, 280 

Posadas, 242 Salimbini, 371 

Pratt, 347, 366 Salmon, 13, 342 

Prazmowski, 286 Sanarelli, 342 

Prior, 367 Sanfelice, 431 

Prowazek, 397, 417 Schafer, 4 : 

Prucha, 180 Schaudinn, 155, 162, 168, 377, 378, 411, 

Prudden, 195 422, 425, 427, 431, 434, 436, 456 * 
Scheele, 6 

Quincke, 264 Schenck, 251 
Schereschewsky, 377 

Rabinowitsch, 47, 327 Schewiakoff, 159, 418, 453 

Ramond, 252 Schick, 309 

Rattone, 290 Schmidt, 106 

Rayer, 10, 281 Schénlein, 247 

Redi, 3 Schottmiiller, 270 

Reed, 376 Schréder, 6 

Reichert, 376 Schiifiner, 443 

Reimarus, 9 Schulze, F., 3, 6 

Remak, 247 Schulze, F. E., 419 

Remlinger, 389 Schiitz, 354 

Rettger, 204 Schwann, 6 

Ricketts, 253, 394 : Schwartz, 261 

Rideal, 77 Schweinitz (De Schweinitz), 392 

Rindfleisch, 11, 271, 275 Sclavo, 285 

Riviére, 376 Sedgwick, 184, 195 

Rivolta, 313 Seguin, 289 

Robin, 246 Sellards, 394 

Rogers, 125, 167 Semmelweiss, 274 

Romanowsky, 42, 155 Sergent, Edm., 414 

Rosenau, 294 Sergent, Et., 414 

Rosenbach, 271, 275 Shiga, 351 

Rosenow, 304 Sholly, 305 


Ross, 264 Siedentopf, 16 


Sihler, 253 
Silberschmidt, 280 
Silbey, 313 

Simon, 385 

Sinton, 408 

Slater, 77 

Smillie, 393 

Smith, Erwin F., 180 
Smith, G. H., 167 
Smith, J. W., 382 


Smith, Theobald, 13, 101, 129, 291, 313, 


356, 448 
Sobernheim, 232, 285 
Solowjew, 455 
Spallanzani, 3, 5, 6 
Starcovici, 448 
Steinhardt, 389 
Stempell, 451 
Stephens, 409 
Sterling, 41 
Sternberg, 266 
Stevens, 409 
Stewart, 37, 38 
Strasburger, 106 
Straus, 356 
Stribolt, 356 
Strickland, 401 
Strong, 340 
Strong, R. P., 377 
Swellengrebel, 401 
Swift, 377 


Takaki, 292, 372 
Taniguchi, 372 
Taylor, 104, 105, 242 
Terre, 313 

Thom, 145, 240 
Thomas, 287 
Thomson, 408 
Tissier, 358 

Todd, 368, 369, 407 
Torrens, 348 
Torrey, 397, 398 
Toussaint, 329 
Trevisan, 329 
Trudeau, 323 


INDEX OF NAMES 463 


Tucker, 194 
Tunnicliff, 257) 358, 378 
Tyndall, 6 


Uhlenhuth, 322, 326 


‘Vallery-Radot, 329, 391 

Van der Brock, 6 

Van Dusch, 6 

Van Ermengem, 53, 294, 295, 362 

Van Leeuwenhoek, 3, 5, 9, 15 

Vaughan, 231, 308, 339, 341 

Veillon, 116, 129, 358 

Vianna, 412 

Vignaud, 285 

Villemin, 320 

Vincent, 256, 357 

Von Behring, 12, 215, 234, 292, 293, 305, 
307 

Von Frisch, 340 

Von Langenbeck, 245 

Von Lingelsheim, 273, 276, 292 

Von Pirquet, 226, 233, 261, 323 


Walker, 348 
Wani, 376 
Ward, 296 
Wasielewski, 437 
Wassermann, 228, 229, 231, 283, 292, 
388 
Washburn, 87 
Webb, 230 
Wechsberg, 222 
Weeks, 309, 310 
Weichselbaum, 262 
Weigert, 59, 214 
Weinberg, 289 
Welch, 278, 287 
Wentworth, 394 
Wenyon, 414 
Wernicke, 242 
Wertheim, 258 
Wheeler, 195 
Whipple, 188 
Whitmore, 418, 419, 422, 425 


. 


eo Se INDEX OF NAMES 


Wilder, 394 

Wilfarth, 14 ; 

Willidms, A. W., 300, 301, 306, 354, 421, 
425 

Williams, H. U., 187, 400 

Williams, W. W., 316 

Williamson, 50, 322 

Wilson, 396 

Winogradsky, 171 

Winslow, 167, 195, 353 

Wolbach, 242, 396 

Wolf, 255 

Wolff-Eisner, 323 

‘Waldhifge, IQI 


Wright, A. E., 16, 26, 27, 218, 224, 235) 
277, 35° 

Wright, J. H., 129, 256, 262, 415 

Wright, Jonathan, 340 


Yersin, 301, 330, 332, 334 
Yorke, 410 ~ 


Zeiss, 16 

Zeller, 454 

Zettnow, 155, 361, 362 
Ziemann, 411, 434 
Zinsser, 265, 345, 353 
Zsigmondi, 16 


INDEX OF SUBJECTS 


(An asterisk (*) designates pages showing illustrations.) 


Abbé condenser, 16, 24,* 30* 
Aberration, chromatic, 15, 20 
spherical, 19 
Abiogenesis, 3, 4 
Abortion, contagious, 356 
_Abrin, 178 
Abscess, 275, 277 
‘Absorption of oxygen for anaerobic 
culture, 129 
Accidental infection, 108 
Acetic acid, 176 
Achorion schénleinii, 10, 247, 249* 
Achromatic objectives, 15 
Acid, carbolic, 76 
Acid-proof bacilli, 47, 313, 328 
method of staining, 47 
Acids, germicidal action of, 71 
antiseptic and preservative action 
of, 79 
Acne, 357 
Acquired immunity, 227, 229 
active, 229 
passive, 231 
Actinomyces, 254 
bovis, 254, 255* 
Actinomycosis, 254 
Active immunity, 229 
duration of, 229 
methods of inducing, 229 
Adaptation to environment, 174, 178 
to parasitism, 209 ; 
. Aédes (Stegomyia) calopus, 375 
Aerobes, 173 
sporogenic, 279 
Aerobic bacteria, 173 
Aerobioscope, 184* 
30 


* 


Agar, 92 
ascitic-fluid, ror 
blood, roz 
blood-streaked, ror 
glucose, 93 
glycerin, 93 
sugar, 93 
Age factor in susceptibility, 204 
Agglomerin, 276 
Agglutination, 218, 348, 353 
Dreyer’s method, 348 
technic of, 218, 348 
Agglutinins, 218 
Ageressins, 212 
Agriculture, 14 
relation of microbes to, 14 
Air, 183 
disease-bearing insects of, 183, 185 
micro-organisms of, 183 
Albumen fixative, 56 
Alcohol, as germicide, 78 
production of, 176 
Alcoholic fermentation, 176 
Aleppo boil, 415 
Alexin, 219, 222 
Alimentary canal, bacteria of, 209, 337 
infection, 138 
Alkalies, germicidal action of, 73° 
Allergy, 226, 233, 322 
Alopécia areata, 250 
Alum in water filtration, 188 
Amboceptor, 221,* 223 
Ameba (Ameeba), 160,* 161, 
cultures of saprophytic, 421, 425 
in tropical dysentery, 12 
American filtration, 188 


465 


466 


Ammonia, 171 
Ameeba, 160,* 161 
‘cultures of saprophytic, 421, 425 
in tropical dysentery, 12 
proteus, 420* 
Amphitrichous bacteria, 154 
Anaerobes, 173 
sporogenic, 286 
Anaerobic bacteria, 173 
cultivation of, 96, 128, 134 
Anaerobic cultures, 128, 134 
Buchner’s method, 129, 130* 
combined hydrogen and pyrogallate 
method, 132,* 133 
deep stab, 128 
fermentation tube, 129 
in eggs, 96 
in hydrogen, 130, 131* 
Novy’s method, 126, 127,* 130, 
131* 
reducing substances in, 134 
removal of oxygen, 129 
under paraffin, 134 
Veillon tube, 129 
Anaphylaxis, 233, 308 
Aniline dyes, 40 
disinfectant action of, 78 
Aniline-water staining solutions, 41 
Animalcules, 9 
Animal experimentation, 135 
value of, 135 
Animals, care of, 135 
experimentation with, 135 
holding of, 136 
inoculation, 137 
observation of infected, 140 
post-mortem examination, 140 
Anopheles mosquitoes, 441,* 442,* 447* 
Anthrax, 10, 12, 281, 283 
bacillus, 281* 
colony, 282* 
immunity, 284 
infection, 284 
intestinal, 284 
pulmonary, 284 
pustule, 284 


INDEX OF SUBJECTS 


, Antharax, serum, 285 

vaccine, 284 
Antiaggressins, 225 
Antibacterial serum, 219, 220 
Antibodies, 215, 225 

distribution of, 225 

source of, 225 
Anticomplementary reaction, 384 
Antiformin method, 50, 322 
Antigen, 224, 235 

for Wassermann test, 384 
Antimeningococcus serum, 263 
Antipneumococcus serum, 268, 270 
Antisepsis, 62, 78 
Antiseptic surgery, 11 
Antiseptics, 78 ‘ 

testing of, 80 
Antistreptococcus serum, 274 
Antitoxic serum, 215 
Antitoxin, diphtheria, 12, 215, 306_ 

concentration of, 306 

curative value of, 308 

preparation of, 306 

prophylactic value, 308 

standardization of, 307 
Antitoxin, tetanus, 12, 215, 292 
Antityphoid vaccination, 350 
Apochromatic objectives, 16 
Arnold steam sterilizer, 67,* 68* 
Artificial culture, 170 
Ascitic fluid, sterile, 99 

agar, IOI | 

collection of, 99 

with sterile tissue, 101 
Ascomycetes, 143 
Asiatic cholera, 360, 363 

carriers of, 366 

diagnosis, 365 

epidemics of, 363 

history of, 363 

prophylaxis, 366 

quarantine in, 366 

spirillum of, 360 

transmission of, 364 

vaccine for, 366 
Aspergillosis, 239 


Aspergillus fumigatus, 239* 
glaucus, 144,* 239 
Atmosphere, bacteria of, 183 
hydrogen, for anaerobes, 130 
Atrichous bacteria, 154 
Attenuation, 209 
Autoclave, 68, 69* 
sterilization, 68 
test objects, 70 
Autopsies, 107, 140 
Avenues of infection, 204 
Avian tuberculosis, 313, 325 
Avoidance of contamination, 108 
Azotobacter, 182 
Azure, methylene, 43, 44 


Babesia, 161, 164,* 448 
bigemina, 448* 
immunity to, 449 
transmission of, 448 
canis, 449 
muris, 164* 
Bacillacer, 279 
Bacilli, 150, 151* 
acid-proof, 47, 313 
capsulated, 52, 154,* 340, 353 
chromogenic, 359 
colon-typhoid, 337 
pigment-producing, 359 
Bacillus, 5, 148, 150 
abortus, 356 
acne, 357 
aerogenes, 339 


aerogenes capsulatus (B. welchii), 


287 
alkaligenes, 342 


anthracis, 10, 12, 281,* 282,* 283* 
anthracis symptomatici (feseri), 287 


avisepticus, 329 
bifidus, 358 


Bordet-Gengou (B. pertussis), 309 


botulinus, 295 
bulgaricus, 358 
butter, 327 
capsulatus, 341 
chancri, 312 


INDEX OF SUBJECTS 467 


Bacillus, chauvei (B. feseri), 287 
cholere-suis (B. suipestifer), 342 

Bacillus coli, 337* 
cultures, 338* 
detection of, 339 
in water supplies, 193 
pathogenic properties, 339 
poisons of, 339 

Bacillus, comma (Sp. cholera), 360 
cyanogenus, 359 

Bacillus diphtheriz, 298, 299,* 300* 
animal inoculation, 298, 301, 305 
bacilli resembling, 302, 305, 309 
cultural characters, 300 * 
granular types, 298, 2997 
in human body, 303 
Léffler’s serum for culture, 300, 

303,* 304 

mode of infection, 305 
morphology, 298 
resistance, 301 
solid types, 298 
‘staining of, 299, 304 
toxin of, 301 
types, 299,* 3007 
virulence, 305 

Bacillus, Ducrey’s, 312 
dysenterie, 351 
edematis, 286 
enteritidis, 341 
fecalis alkaligenes, 342 

' feseri, 287 

fluorescens, 359 
fusiformis, 357 
Gartner’s (B. enteritidis), 341 
gas (B. welchii), 287 
grass (B. midlleri), 327 
hay (B. subtilis), 280*. 
hoffmanni, 309 
icteroides, 342 
influenze, 311 
Klebs-Loffler (B. raat , 298 
Koch-Weeks, 309, 310* 
lactici-acidi, 197 
lactis aerogenes (B. aerogenes), 339 
leprae, 326 


468 


4 


e 


INDEX OF SUBJECTS 


Bacillus, mallef, 354* 


melitensis, 334 
mesentericus, 279 

milleri, 327 
Morax-Axenfeld, 309, 310* 
mucosus, 341 
murisepticus, 330 
mycoides, 279 

ozen®, 341 

paracolon, 343 
paradysentery, 352 
paratyphoid, 342 
perfringens (B. welchii), 287, 288* 
pertussis, 309 


Bacillus pestis, 330* 


cultures, 331 
immunity, 333 

in animals, 332 
morphology, 330* 
toxins, 331 


Bacillus plurisepticus, 330 


pneumoniz, 340* 

potato (B. vulgatus), 279 

prodigiosus, 359 

proteus, 358 

pseudo-diphtheria (B.' hoffmanni), 
309 

psittacosis, 342 

pyocyaneus, 359 

radicicola, 14 

rhinoscleromatis, 340 

rhusiopathize suis, 330 

salmonii, 342 

Shiga’s (B. dysenteriz), 351 

smegmatis, 327 

subtilis, 280* 

suipestifer, 342 

tetani, 290 


Bacillus tuberculosis, 313, 314* 


amphibian, 326 
avian, 325 
bovine, 324 

_ branching of, 316* 
chemical composition of, 316 
cultures of, 315, 317* 
fish type, 326 


Bacillus tuberculosis, human 
314,* 315* 
morphology, 314 
poisons of, 317 
resistance of, 318 
varieties of, 313 
Bacillus typhi murium, 342 
Bacillus typhosus, 343,* 344* 
agglutination of, 348 
distribution of, 343, 346 
flies as carriers of, 350 
human carriers of, 346, 350 
in blood, 346 
in feces, 347 
in food, 349, 35° 
in milk, 350 
in soil, 350 
in sputum, 346 
in urine, 346 
. in water, 194, 349 
isolation of, 347 
pathogenic properties of, 343, 345 
poisons of, 345 
resistance of, 345 
vaccines, 350 
Bacillus violaceus, 359 
vulgaris, 358 
vulgatus, 279 
welchii, 287, 288* 
xerosis, 309 
Bacteremia, 207 
streptococcus, 274 
Bacteria, 147 
acid-proof, 47, 313 
adaptability, 175 
aerobic, 173 
anaerobic, 173, 286 
classification, 143, 147, 166, 167 
colonies, 113,* 115, 179 
cylindrical, 150 
dimensions, 147 
discovery, 3 
distribution, 181 
fluctuation, 174 
food, 196 
in air, 181, 183 


type, 


INDEX OF 
Bacteria, in agriculture, 14, 182 
in food, 196 
in ice, 185 
in milk, 196 
in soil, 182 
in water, 185 
soil, 182 
spherical, 148* 
spiral, 152, 153* 
structure of, 153 
variation, 175 
with spores, 155* 
Bacteriaceze, 150 
Bacterial poisons, 178, 210 
vaccines, 12, 229, 230 
Bactericidal substances, 219, 234 
Bacteriology, 1 
biological relations, 3 
history, 1 
hygienic, 7 
nomenclature of, 167 
scope, 2 
Bacteriolysins, 220 
Bacteriolysis, 220 
Bacterium, 5, 150 
Balantidium coli, 166, 454, 455* 
parasitic relations, 455 
pathogenesis, 455° 
Balantidium minutum, 456 
Barber’s itch, 250 
Basic dyes, 40 
Basidiomycetes, 145 
Basophile granules, 60 
Bed-bugs (Cimex), 413 
Beef-tea (broth), 84 
Berkefeld filter, 63 
Bichloride of mercury, 74 
Biological relationships of bacteriology, 


3 
Birds, malaria of, 431, 433, 434, 436 
trypanosomes of, 409, 410 
tuberculosis of, 313, 325 
Black death (plague), 332 
Black-leg, 287 \ 
Blastomyces dermatidis, 253 
Blastomycetes, 146,* 252 


SUBJECTS 469. 
Blastomycetic dermatitis, 253 
Blastomycosis, 252 
Bleaching powder, 73 
Blepharoplast, 397 
Blood-agar, Novy’s, 101 
Pfeiffer’s, 1o1 
Blood, 95, 97 
bacteria in, 207 
citrated, 98, 105 
culture, 104 
defibrinated, 98 
films for microscopic examination, 
55 
pipette for collection of, 98,* 99, 
105* 
protozoa in, 207 
sterile, collection of, 97 
Blood serum, 95 
as culture medium, 96. 
Léeffler’s, 96 
Blue milk, 359 
Blue pus, 359 
Bodo lacerte, 417* 


* 


Boil, Delhi, 415* 


Boils, 277 
Boéphilus bovis, 165, 448 
Bordet-Gengou bacillus (B. pertussis), 
309 
Boric acid, 79 
Botrytis bassiana, 10, 243 
Botulin, 296 
Botulism, 296 
; antitoxin for, 296 
Bouillon (broth), 84 
Bovine pleuro-pneumonia, 388 
tuberculosis, 324 
Branching bacilli, 316 
Bread-paste, 97 
Bromine as a germicide, 74 
Broth, nutrient, 84 
containing sterile tissue, 101 
sugar, 93 
sugar-free, 92 
Brownian movement, 35 
Bubonic plague, 332, 333, 334 
diagnosis, 331 


470 


Bubonic plague, fleas as carriers of, 333 
history of, 332 : 
immunity to, 333 
prophylaxis of, 334 
rodents as reservoirs of, 332, 333 
serum, 334 
vaccines, 333 
Buchner method for anaerobic culture, 
129, 130* 
Burner, Bunsen, 109 % 
_ Koch’s automatic safety, 123,* 
Butter bacillus, 327 
Butyric acid test, 264 


124 


Calcium oxide (lime), 73 
Calmette’s test (tuberculin), 323 
Capsules, 154* 
staining of, 52 
Carbol-fuchsin, 41 
Carbol-gentian-violet, 41 
Carbolic acid, as a germicide, 76 
Carbuncles, 277 
Carmine, 61 
Carriers of infection, 208 
Caseation, 320 
Cattle plague, 388 
Cattle tick, 165, 448 ‘ 
Cedar-wood oil, 30 
Cell, chemical constitution of, 214 
Cell-membrane of bacteria, 153 
‘Celloidin, 55 
Cell-receptors, 216,* 217,* 221* 
Cerebro-spinal fluid, 264 
collection of, 105, 264 
examination of, 264 
in meningitis, 264 
in tuberculosis, 322 
test for globulins in, 264 
Chancroid, 312 
Charbon (Anthrax), 281, 283 
Chart, descriptive, 180 
Cheese, 188, 240, 244 
Chemical agents as germicides, 71 
disinfection, 72, 77 
effects, 175 
products, 175 


INDEX OF SUBJECTS 


Chicken cholera, 329 
sarcoma, 306 
Chlorine, as a germicide, 73 
Chloroform, preservative action of, 79 
Cholera, Asiatic, 360, 363 
carriers, 366 ‘ 
diagnosis of, 366 
prophylaxis, 366 
transmission of, 364 
Cholera, fowl, 329 
Cholera, hog, 392 
Chromatic aberration, 15, 20 
Chromatin, 155 
Chromogenic bacteria, 359 
Ciliates, 166, 452* 
Ciliophora, 157, 166, 452* 
Cladothrix, 257, / 
Classification, 4, 5, 143, 166, ee 
of molds, 143 : 
of protozoa, 156 
of yeasts, 143 
outline of micro-organisms, 166 
Claviceps purpurea, 240 
Cleaning fluid, 37, 84 
Clearing microscopic preparations, 28° 
Clostridium, 152,* 286 
botulinum, 294,* 295 
edematis, 286 
feseri, 287 
periringens, 287, 288* 
tetani, 290 
Coccacee, 148,* 258 
Cocci, 148,* 258 
Coccidioidal granuloma, 242, 243 
Coccidioides immitis, 242* 
Coccidiosis, 430 
Coccidium (Eimeria), 161, 162* 
cuniculi (Eimeria steidz), 429,* 
430* 
Coccus, 148* 
Cold, effect on bacteria, 64 
antiseptic action of, 79 
Collection of material, 103 
of sterile ascitic fluid, 99 
of sterile blood, 97, 104 
of sterile tissue, 100 


INDEX OF 


Collodion capsules, 138, 139* 
embedding, 55 
Colon bacillus, 337* 
cultures of, 338* 
detection of, 339 
in water-supplies, 193 
pathogenic properties of, 339 
poisons of, 339 
Colonies of bacteria, 110, 113,* 115,* 117 
Comma bacillus, 360 
Commensal, 201 
Complement, 222, 382 
deviation of, 222 
detection of, 224 
fixation of, 223, 382 
Complement-fixation test, 382 
antigen, 384 : 
blood cells for, 382 
complement for, 382 
hemolytic amboceptor for, 382 
patient’s serum for, 383 
signification of, 387 
technic of, 386 
Condenser, sub-stage (Abbé), 24,* 30* 
dark field, 25* 
Conjunctivitis, 309, 310* 
Conorhinus megistus, 413, 414* 
as vector of coreotrypanosis, 413 
Consumption (tuberculosis), 313 
Contagion, 7, 8, 9, 207 
early ideas of, 7, 8, 9 
Contagious abortion, 356 
_ digease, 207 
Contamination, avoidance of, 108 
Coreotrypanosis, 411 
transmission of, 413 
Cornet forceps, 38* 
Corrosive sublimate as a germicide, 74 
Cotton plugs, 84 
Cover-glass forceps, 37, 38* 
preparations, 38, 44 
. Cover-glasses, 37 
cleaning of, 37 
Cow-pox, 12 230, 395 
Creolin, 77 
Cresol, 77 


SUBJECTS 471 


Croupous pneumonia, 268 
Cryptococcus gilchristi, 252* 
Culex mosquitoes, 446, 447,* 
Cultivation, 108 

of anzrobes, 128 

of bacteria, 108 

of protozoa, 13, 101, 400, 402, 411, 

413, 415; 421, 425 

of spirochetes, 13, 369 
Culture media, 8 

agar, 92 

bread-paste, 97 

broth, 84 

blood-agar, tox 

blood-serum, 95 

choice of, 119 

containing uncooked protein, 97 

dextrose, 93 

dextrose-free, 92 

Dorset’s egg, 97 

Dunham’s solution, 94 

filling into tubes, 91* 

gelatin, 90 

lactose, 93 

litmus, 93 

Léffler’s blood serum, 96 

method of inoculating, 117* 

milk, 94 

modified, 92 

nitrate-broth, 95 

peptone solution, 94 

potato, 93 

preparation of, 84 

special, 93 

titration of, 85, 87 
Cultures plate, 12, 110, 113* 

anzrobic, 128 

pure, 117 

roll-tube, 114,* 115* 

sealing of, 119 

smear, 116, 117, 118* 

stab, 118,* 128 

stock, 118 

streak, 116, 117, 118* 

tube, 111, 116, 118,* 129 
Cutaneous tuberculin test, 323 


476 , 


Immunity, antiaggressive, 225 
antitoxic, 232 
bacteriolytic, 219 
combined passive and active, 232 
duration of, 229 
Ehrlich’s theory of, 234 
following vaccination, 229 
individual, 228 
Immunity, mechanisms of, 232 
natural, 227 
of species, 227 
passive, 233 
racial, 228 
theories of, 234 
unit, 293, 3¢7 
Immunology, 235 
Impression preparation, 38 
Inactivated serum, 220, 383 
Incubator, 119, 120* 
low temperature, 125, 126,* 127, 128 
rooms, 124 
Infantile paralysis, 393, 304 
Infection, 7, 203 
avenues, 204 
general, 206 
healthy carriers of, 208 
local, 206 
possibility of, 203 
secondary, 206 
transmission of, 204, 207 
Infectious disease, 203, 213 
facts and theories of, 213 
phenomena of, 213 
Infectious jaundice, 373 
Influenza, 311 
Inoculation, animal, 137, 138 
into the circulating blood, 137 
into the cranial cavity, 137 
intracardiac, 138 : 
intraperitoneal, 137 
subcutaneous, 137, 138 
Inoculation of culture media, 111* 
Inorganic salts as microbic food, 172 
as germicides, 74 
Insects, 13 
destruction of, 72 


‘ INDEX OF SUBJECTS 


Instruments, sterilization of, 66* 
Intermediary body, 221* 
Intermittent sterilization, 70 
Intestinal amebe (endamebe), 421* 
anthrax, 281 
juice, collection of, 106 
Intestine, infection through, 205 
Intrauterine infection, 204 : 
Intravenous inoculation, 137 
Invisible microbes, 9, 26, 156, 388 
Iodide of mercury, 75 
Todine as a germicide, 74 
antiseptic value, 79 


_ Iodoform, 74 


Iris diaphragm, 25. 

Iron hematoxylin, 54 

Isolation of bacteria, 109 
plate method, 110 
streak method, 116 
Veillon method, 116 

Itch (scabies), 9 


Jaw, lumpy (actinomycosis), 254 
Jeffer’s plate, 191* 

Jennerian vaccination, 230, 395 
Jenner’s stain, 42 


Kala-azar, 413, 415 
parasite of, 413 
transmission of, 414* 
Kefir, 199 
Kirkbride forceps, 39* 
Klebs-Léffler bacillus, 298 
Koch-Eberth bacillus, 343* 344* 
Koch’s safety burner, 123,* 124 
plate cultures, r10 
postulates, 202 
steam sterilizer, 66* 
Koch-Weeks bacillus, 309, 310% 
Koumiss, 199 


Lactic acid, 176 

Lactobacillus, 358 
bifidus, 358 
bulgaricus, 358 


as oes 


INDEX OF SUBJECTS 


Lamblia, 159 
intestinalis, 159,* 418,* 419 
Leishman-Donovan bodies (L. dono- 
vani), 158, 159,* 413 
Leishmania donovani, 158,* 159,* 413, 
414,* 415* 
cultures, 159,* 413 
occurrence, 413 
transmission, 413 
Leishmania infantum, 416 
Leishmania tropica, 415,* 416* 
cultures of, 415, 416 
immunity of, 416 
Leishman’s stain, 42 
Leprosy, 326, 327 
Leptomonas culicis, 397 
Leptospira, 373 
ictero-hemorragiz, 373* 
Lepfothrix, 257 
buccalis, 257 S, 
Leukocidin, 276 
Leukocytozoén (Hemoproteus), 434” 
lovati, 435,* 436 
Levaditi’s silver stain, 379, 3807 
Ligatures, sterilization of, 81 
Light, effect on bacteria, 63 
Lime, disinfectant action of, 73 
Lithium carmine, 61 
Litmus, 85, 87 
Lockjaw (tetanus), 290, 291 
Locomofion, 35 
Léffler’s bacillus (B. diphtherie), 2098, 
299,* 300* 
blood serum, 96 
flagella stain, 52 
methylene blue, 42 
Lophotrichous bacteria, 154* 
Lower bacteria, 148, 153 
Low-temperature incubator, 125, 126* 
Luetin, 379 
test, 386 
Lumpy jaw, 254 
Lungs, infection of, 205 
inflammation of (pneumonia), 268 


’ Lysins, 219 


Lysol, 77 


477 


Lyssa (rabies), 389, 391 
diagnosis of, 391 
Hogyes treatment of, 230 
Pasteur treatment of, 391 


Macrogametes, 162,* 163 
Macrogametocytes, 162,* 163 
Madura foot, 256 
Madurella mycetori, 256 
Magnification, 16,* 17,* 18,* 19, 23 
Malachite green, 78 , 
Malaria, 444 
avian, 431, 433, 434, 436 
diagnosis of, 446 
estivo-autumnal, 438, 445 
mosquitoes in, 446, 447* 
prophylaxis, 446 . 
quartan, 444, 445* 
tertian, 442, 443,* 445 
transmission of, 446 
Malarial parasites of birds, 431, 433, 434 
436 
of man, 438, 439,* 440,* 441;* 442," 
443," 444,* 445* 
of monkeys, 448 
transmission of, 438, 446 
Mal de Cadéras, 407 
Malignant edema, 287 
'  -pustule, 284 
Mallein, 355 
Malta fever, 334, 335 
diagnosis of, 335 
Mammalian tuberculosis, 313, 314,* 
315,* 324 
Marmorek’s serum (antistreptococcus 
serum), 274 
Mastigamceba aspera, 41 8,* 419 
Mastigophora, 157, 397 
Mayer’s glycerin-albumen, 56 
Measles, 394 
Mechanical filtration, 188 
sterilization, 62 
Media, culture, 83 
Mediterranean fever (Malta fever), 334, 
335 
Membranous croup (diphtheria), 302 


28 


482 INDEX OF 
Rabies, 389, 390 
diagnosis of, 391 
Hégyes treatment of, 230 
Negri bodies in, 389, 390* 
Pasteur treatment, 391 
treatment of wound, 391 
Racial immunity, 228 
Radiolaria, 426 
Rat-bite fever, 372 
Rats, relation to bubonic plague, ‘332, 
333, 334 
relation to Weil’s disease, 373 
trypanosomes of, 400 ; 
Rauschbrand (symptomatic anthrax) ,287 
Ray fungus (actinomyces), 254, 255* 
Reaction, cutaneous, 323 
of culture media, 86, 172 
of host to infection, 213 
Reading glass, 19 
Receptor of first order, 216* 
of second order, 217* 
of third order, 221* 
theory of immunity, 234 
Reducing substances, 134 
Regulation of temperature, IIg 
Regulator, electric, 126* 
Roger’s, 125, 126* 
Regulator, gas, 120 
MacNeal, 121, 122* 
method of filling, 122 
Reichert, 121* 
Roux, 123* 
Relapsing fever, 11, 368 
diagnosis, 370 
spirochetes, 368 
Resistance to infection, 203, 228 
Respiratory infection, 138 
Rhinoscleroma, 340 
Rhipicephalus annulatus, 165, 448 
Rhizopoda, 161, 420, 426 
Ricin, 178 
Rinderpest, 230, 388 
Roll-tubes, 114,* 115* 
Romanowsky stain, 42, 44, 155 
Rooms, disinfection of, 71, 77 
incubator, 119, 120,* 124 


SUBJECTS 


Root-tubercle bacteria, 14,182, 201 
Rubber caps, 118,* 119,124 
stoppers, 118,* 119, 124 


Rules of Koch, 202 


Russell’s medium, 348 


Saccharomyces, oe ce 
‘cervisie, 146,* 
ellipsoideus, 46," 
Sanarelli’s bacillus (Bacillus icteroides), 
342 
Sand filtration, 187 
Saprogenic bacteria, 177 
Saprophyte, 171 
Saprophytic, 171 
Sarcina, 148,* 149 
aurantiaca, 278 
ventriculi, 278 
Sarcoma, chicken, 396 
Sarcoptes scabei, 9 
Schick reaction, 309 
Schizomycetes, 147 
Schizotrypanum cruzi, 411, 412,* 413 
cultures of, 413 
transmission of, 413 
Sealing culture tubes, 118,* 119, 124 
Secondary infection, 206 
Section-cutting, 57 
Sections, 58 
staining of, 58, 59 
tubercle bacilli in, 60 
Sedgwick-Tucker aérobioscope, 184* 
Sedimentation, 63 
Self-purification of water, 186 
Semen, transmission of infection by, 205 
Sensitizer, 221 
Septicemia, 11, 207 ‘ 
hemorrhagic, 329 
sputum, 266 
Serum, anthrax, 285 
antibacterial, 219 
antimeningococcus, 263 
antipneumococcus, 268, 270 
antistreptococcus, 274 
antitoxic, 215 
bactericidal, 219 


* 


INDEX OF 


Serum, blood, 95. 
cytolytic, 220 
dysentery, 352 
hemolytic, 220 
immune, 320 | 
Léffler’s, 96 
normal, 219 
plague, 334 
Pneumococcus, 268, 270 
sterilizer for, 96*. , 
Yersin’s, 334 fem bak 
Shiga’s bacillus (Bacillus dysenteriz), 
35t 
Side-chain theory, 214, 234 
, Silver nitrate as a germicide, 76 
Sleeping sickness, 407, 409 
transmission of, 407 
trypanosome of, 407 : 
tsetse fly concerned in, 407, 408* 
Slides, forceps for, 39 
glass, 39 
method of cleaning, 39 
Small-pox, 395 
inoculation, 12, 229 
vaccination, 12, 230, 395 
virus of, 395 
Smear culture, 117, 118* 
Smear preparations, 37 
on cover-glass, 37 
on slide, 39 
Smegma bacilli, 327 
Soaps, germicidal action of, 71 
Sodium hydroxide, normal solution of, 
“a: 
Sodoku, 372 
Soft chancre, 312 
Soil bacteria, 182, 183 
Solutions, normal, 85 
Soor (thrush), 10, 245 
Sore, Oriental (Delhi boil), 415* 
Souring of milk, 198 
Species of bacteria, 174 
stability, 174 
variation, 174 
Specific nomenclature, 167 
Spherophrya pusilla, 456* 


SUBJECTS 


Spherical aberration, 19 
Spherical bacteria, 148,* 149 
Spinal fluid, 264, 
Spirilla, 148, 152, 153* 
Spirillacere, 152, 360 
Spirillum, 5, 148, 152, 153* 
Spirillum cholerz, 360, 361,* 
agglutination, 365 
cultures of, 360 
immunity, 363, 366 
in feces, 365 
in water, 306 
poisons of, 363 
resistance of, 361 
transmission of, 364 
Spirillum, Deneke’s, 367 
metchnikovi, 367 
of Finkler and Prior, 367 
rubrum, 360 
tyrogenum, 367 
Spirocheta, 5, 13, 152, 153, 368 
anserina, 371 
culture of, 13, 360 
duttoni, 369 
fusiformis, 357 
gallica, 376 
gallinarum, 371 
hebdomadalis, 376 
ictero-hemorrhagie, 373* 
icteroides, 374* 
kochi, 369 
microdentium, 387 
muris, 371, 372* 
novyi, 369, 370* 
obermeieri, 368 
of relapsing fever, 368 
Spirocheta pallida, 377, 378,* 380* 
animal inoculation of, 379 
antibodies, 381 
cultures of, 377, 379 
in blood, 382 
— microscopic demonstration 
381 
morphology, 377. - 
staining of, 378 
Spirocheta plicatilis, 368 


483 


of, 


478 INDEX OF 


Meningitis, 262 
diagnosis, 264 
serum, 263 
serum treatment, 264 
Meningococcus, 262, 265* 
cultures of, 262 
Mercuric chloride as a germicide, 74 
iodide, 75 


Metchnikoff’s phagocytic theory, 234, 


235 

Methyl violet, 78 
Methylene azure, 43, 44 
Methylene blue, 40, 42 

germicidal power of, 78 
Methylene violet, 44 
Miasm, 8, 207 
Microaérophilic microbes, 173 
Microbe, 3 2 

relation of, to environment, 178 
Microbic variation, 174 - 
Microbiology, 3 
Micrococcus, 5, 148,* 149 

agilis, 278 

catarrhalis, 265 

gonorrhes; 258, 259* 

melitensis, 334 

meningitidis, 262, 265* 

tetragenus, 278 
Microgamete, 162,* 163 
Microgametocyte, 162,* 163 
Micromillimeter, 31 
Micron, 31 
Micronucleus, 452 
Micro-organisms, 3 

distribution of, 181 

in air, 183 

in food, 196, 200 

in ice, 195 

in milk, 196 

in soil, 182 

in water, 185 
Microscope, 15, 21,* 29* 

dark-field, 25,* 36 

development of, 15 

eye-pieces, 22,* 30 

objectives, 20,* 22, 30 


SUBJECTS 


Microscope, principle of, 16, 22* 
tandem, 16 
use of, 31 
Microscopic definition, 24 
measurements, 31 
resolution in depth, 24 
Microspira, 152 


Microspira comma (Sp. cholere), 360 


Microsporon audouini, 250 - 
furfur, 250 * 
septicum, I1, 271 

Microtome, 57* 


Migula’s classification of bacteria, 148 


Miliary tuberculosis, 321 
Milk, 196 
acid, beverages, 358 
as culture medium, 94 
bacteria of, 196 
blue, 359 
collection of samples of, 103 
composition of, 196 . 
for infant feeding, 199 
pasteurization of, 198 
micro-organisms of, 196 
Milzbrand (anthrax), 281, 283 
Mixed infection, 206 
Moist heat, effect on bacteria, 65 
Modes of entry of infection, 204 


s 


Moisture requirement of bacteria, 171 


Molds, 143, 144,* 237 


Miéller’s grass bacillus (Bacillus mélleri), 


327 
spore stain, 51 
Monilia candida, .245,* 246* 
psilosis, 247 
Monotrichous bacteria, 154* 
Morax-Axenfeld bacillus, 309, 310* 
Morphology, 143 
relation of, to environment, 178 
relation of, to physiology, 169 


Mosquitoes in malaria, 441,* 442, 


447* 
Motility, 35 
Movement, 35 

Brownian, 35 
real, 35 


* 


INDEX OF 


Mucor,237, 238* 
corymbifer, 237, 238* 
mucedo, 144,* 237, 238* 

Muscardine, ro, 243 

Musgrave and Clegg’s 

ameba, 421 

Mycelium, 143 

Mycetoma, 256 

Mycetozoa, 426 


medium for 


Nagana, 403, 405 
diagnosis of, 406 
immunity to, 406 
occurrence of, 405 
transmission of, 405 
trypanosome of, 4037 
Nanukayami, 376 
Natural immunity, 227 
individual, 228 
mechanisms, 232 
of species, 227 . 
racial, 228 
Negri bodies, 389, 390* 
Neisseria, 258 
Neisser’s gonococcus, 258 
Neisser-Wechsberg phenomenon, 222, 
223* 
Neosporidia, 165, 449 
Neutralization of culture media, 85, 87 
Nitrate broth, 95 
Nitrate of silver, 76 
Nitrates, production of, by bacteria, 182 
Nitrification, 182 
Nitrifying bacteria, 182 
Nitrites, formation of, 182 
Nitrogen fixation, 171, 182 
Nitrosomonas, 171 
Nocht-Romanowsky stain, 42 
Nodule bacteria (root tubercles), 182, 201 
Nomenclature, 167 
Normal solution, 85 
Nosema, 161, 165* 
bombycis, 10, 165,* 449, 451* 
Novy’s anzrobic method, 130, 131* 
blood-agar, 101 
cover-glass forceps, 38* 


SUBJECTS 479 
Nuclear stains, 61 
Nucleus of bacteria, 155 
- of protozoa, 157 

Number of bacteria in milk, 197 

in water, 190 

required to infect, 204 
Numerical aperture, 23 
Nutrient agar, 92 

broth, 84 

gelatin, 90 


Obermeier’s spirillum, 368, 369* 
Objectives, achromatic, 15, 20,* 30 
apochromatic, 16, 20 
defects of, 20 
immersion, 30 
Ocular tuberculin reaction, 323 
Oculars, 23, 30 
Oidiomycosis, 252* 
Oidium albicans, 10, 245,* 246* 
\ lactis, 144,* 244* 
Oil, aniline, in stains, 41 
Odkinete, 164 
Odspore, 143 
Opalina ranarum, 454* 
Opsonins, 224 
Organic poisons as germicides, 76 
food requirements, 171 
Oriental sore (Delhi boil), 415 
Osteomyelitis, 275, 277 
Outline classification, 166 
Ovum, infection of, 204 
Oxidizing agents as germicides, 73 
Oxygen, 173 
requirement, 173 _. 
removal of, 129 
Oysters as source of typhoid, 350 


Panophthalmitis, 280 
Paracolon bacilli, 342, 343 
Paradysentery bacilli, 352 
Paraffin imbedding, 56 
Paralysis, infantile, 393 
Paramecium aurelia, 453 
bursaria, 453 
caudatum, 452* 


480 -INDEX “OF SUBJECTS 


Paramecium aurelia, conjugation, 452 Phenomena of disease, ‘202 


division, 452 Phenomenon, Pfeiffer’s, 219, 363 
form and structure, 452 Phlebotomus fever, 393 
Paramecium putrinum, 453* Phosphorescence, 175 
Parasite, 172 ‘Photogenic bacteria, 175 
obligate, 172 : Phycomycetes, 143 
‘ Parasitism, 201, 209 Physical sterilization, 62 
Paratyphoid bacilli, 342 Physiological method, 169, 170 
Parenteral digestion, 219 hyperplasia, 213 
Passive immunity, 231 tests, 180 
Pasteur pipettes, 33 Physiology of micro-organisms, 169 
treatment for rabies, 391 relation to morphology, 169 
Pasteur-Chamberland filter, 63 Pipettes, glass (Pasteur pipette), 33* 
Pasteurella, 329 for drawing blood from animal, 99* 
cholere-gallinarum, 329 for drawing blood from man, 98,* 
pestis, 330 105* 
pluriseptica, 330 Piroplasma (Babesia), 161, 164* 
Pasteurization, 66 bigeminum, 448* 
Pathogenesis, 202, 209 canis, 449 
Pathogenic bacteria, 202, 209 muris, 164* 
organisms, 202, 209 Pityriasis, 250 
protozoa, 12, 13, 397 Placental transmission, 205 
soil bacteria, 183 Plague, 330, 332 
Pathology, relation of bacteriology to, 7 bubonic, 333 
Pearl disease (bovine tuberculosis), 324 diagnosis, 331 
Pébrine, 10, 165, 449 fleas as carriers of, 332 
parasite of, 165,* 449, 451* Haffkine’s prophylactic, 333 
restriction of, 451 immunity, 333 
Pediculus humanus, 377, 394 in animals, 332 
Penicillium crustaceum, 240* pneumonic, 333 
glaucum, 144,* 240* prophylaxis of, 334 
rocqueforti, 240 serum, 334 
Peptone solution, 94 transmission, 332 
Peptonizing ferments, 177 Plague vaccines, 333 
Peritrichous bacteria, 154 Planococcus, 149 
Perlsucht (bovine tuberculosis), 324 agilis, 278 
Permanganate of potassium as a germ- Planosarcina, 149 
cide, 74 Plants, diseases of, 241 
Peroxide of hydrogen as a gérmicide, 74 Plasmodium, 12, 161, 163* 
Pertussis, 309 : brassicez, 426 
Petri dishes, 112* Plasmodium falciparum, 163,* 438, 440* 
Pfeiffer’s phenomenon, 219, 363 asexual cycle in man, 439* 
Phagocytic theory, 224, 234 cultures of, 442 
Phagocytosis, 214, 224 pathogenic relation of, 445 
Phenol, 76 sexual cycle in anopheles, 441* 


Phenolphthalein, 86 transmission of, 442, 446 


INDEX OF SUBJECTS 481 


Plasmodium kochi, 448 Products of bacteria, 176 
malarise, 444, 4457 primary, 176 
preecox, 436,* 437,* 438* ptomaines, 177 
vivax, 442, 443,* 444* secondary, 176 
Plasmodroma, 157 toxins, 178 
Plasmolysis, 153 Protective inoculation for anthrax, 285 
Plate cultures, 12, 110 ; for cholera, 366 
Koch’s original method, 116 for diphtheria, 308 
Platinum wire, 32°. for plague, 333, 334 
Pleuro-pneumonia of cattle, 13,388 == ° for small-pox, 12, 395 
filterable virus of, 388 for typhoid, 350 
Plugs, cotton, 84 Proteolysins, 225 
Pneumococcus, 266, 267* Proteolytic ferments, 177 
immunity to, 268, 270 Proteosoma precox, 436,* 437,* 438* 
mucosus, 269 ' | development in blood, 437* 
poisons of, 268 development in the mosquito, 438* 
typing, 269 Proteus vulgaris (Bacillus proteus), 358 
Pneumonia, 268 Protista, 166 
micro-organisms in, 268 Protozoa, 12, 156, 397 
serum, 268,270 relation to disease, 12 
Poisoning, food, 200, 295, 341, 343 wet fixation of, 54 
botulism, 295 Pseudo-diphtheria bacillus, 305, 309 
enteritidis type, 341 Pseudomonas, 150 
proteus vulgaris as cause of, 358 pyocyanea, 359 
Poisons, 200, 210 radicicola, 182, 201 
Poliomyelitis, 393 syncyanea, 359 
‘ Porcelain filter, 63 Ptomain, 177 
Post-mortem examination, 107, 140 Puerperal fever, 273, 274 
Postulates of Henle, 10, 12 Pulmonary anthrax, 284 
of Koch, 12, 202 Pure culture, 117 
Potassium permanganate, 74 Purification of water, 186, 187, 188, 189 
Potato cultures, 93 Pus, collection of, 106 
bacillus (B. vulgatus), 279 Pustule, malignant, 284 
medium, 93* Putrefaction, 5, 177 
Precipitation test, 216 Putrefactive alkaloids, 177 
Precipitinogen, 217 ‘ bacteria, 177 
Precipitins, 216 products, 177 
Predisposition, 204, 206 Pyemia, 207 
Preservation, 6, 62, 79 Pyoktanin, 78 
Preservatives, 79 Pyrogallic-acid anerobic method,129,132 
Pressure, effect on bacteria, 63 Pyrosoma (see Babesia), 448 . 
filter, 63 5 
Products of bacteria, 175 Quartan malaria, 444, 445* 
chemical effects, 175 Quarter evil (symptomatic anthrax), 287 
enzymes, 176 : Quincke’s puncture, 264 
physical effects, 175 - Quotidian malaria, 445 


31 


486 INDEX OF 
Thermogenic bacteria, 175 
Thermostat (thermoregulator), 119, 
121,* 122,* 123,* 126* 
Thrush, 10, 245 
Tick, cattle, 448 1 
Tinea, 250 
versicolor (pityriasis), 250 
Tissues, examination of, 55 
for culture media, 100 
Titration of culture media, 85, 87 
Tongue, wooden (actinomycosis), 254 
Torula, 147 — 
Toxemia, 207 
Toxin, 178, 210 
chemical nature of, 178 
diphtheria, 301 
extracellular, 210 
intracellular, 211 
soluble, 210 
standardization of, 293, 294 
tetanus, 291 
Toxoid, 211 
Toxophore, 211 
Transmission of disease, 13, 204, 207 
Transudates, collection of, 99 
Trench fever, 376 
Treponema pallidum (Spirocheta pal- 
lida), 377, 378,* 380* 
Trichobacteria, 147, 254 
Trichomonas, 159,* 160, 417,* 419 
hominis, 417,* 419 
Trichomycetes, 254 
Tricophyton, 250 
Trimastigamceba philippinensis, - 418,* 
419 
Tropical dysentery, 423 
malaria, 438, 445 
splenomegaly (kala-azar), 413, 415 
ulcer, 415 
Trypanoplasma borreli, 417 
cyprini, 416,* 417 
guernei, 417 
Trypanosoma, 158,* 159, 398 
Trypanosoma avium, 410,* 411 
cultures of, 411 
occurrence of, 410 


SUBJECTS 


Trypanosoma brucei, 158,* 403* 
cultures of, 403, 405 
form and structure, 403, 404 
immunity to, 406 
multiplication of, 405 
occurrence, 405 
poisons of, 405 
transmission of, 405 : 
Trypanosoma equinum, 158,* 403,* 407 
* equiperdum, 158,* 403,* 406* 
evansi, 158,* 403,* 406 
Trypanosoma gambiense, 158,* 403,* 
407 
cultures of, 408 
form and structure, 407 
in animals, 408 
in man, 408 
in the fly, 407 
transmission of, 407 
Trypanosoma lewisi, 158,* 400,* 4or,* 
“ 402,* 403* 
cultures of, 402 
division of, 401 
occurrence of, 400 
Trypanosoma rhodesiense, 409 
rotatorium, 398, 399* 
Trypanosomes, 158,* 398, 403* 
Tsetse-fly (Glossina morsitans), 404,* 
405 
disease (Nagana), 405 
Tubercle, 320 
Tubercle bacillus, 313, 314* 
amphibian, 326 
avian, 325 
bovine, 324 
branching of, 316* 
chemical composition of, 316 
cultures of, 315, 317* 
fish type, 326 
human type, 314* 
in sections, 60 
poisons of, 317 
resistance of, 318 
stain for, 47, 49, 60, 315 
transmission of, 321 
varieties of, 313 


INDEX OF 


Tuberculin, 318 

reaction, 322, 325 

test, 322, 323, 325 

treatment, 323 
Tuberculosis, 319 

avian, 325 

bacillus of, 313, 314* 

bovine, 324 

diagnosis of, 321 

fowl, 325 = 

immunity, 324 

mammalian, 319 

mode of infection in, 321 

tuberculin test in, 322, 323, 325 

tuberculin treatment in, 323 
Type determination of meningococcus, 

263 

of pneumococcus, 269 
Typhoid bacillus (B. typhosus), 343 

carriers, 346, 349 

detection in water, 194 
Typhoid fever, 343, 346 

diagnosis of, 346 

immunity to, 350 

in animals, 345 

prophylaxis of, 350 

transmission of, 349 

vaccines, 350 

vaccination, 350 
Typhus fever, '394 


Udder, bacteria in, 196 
Ulcer, tropical, 415 
Ultramicroscope, 16 
Ultramicroscopic organisms, 156, 388 
Unit, immunity, 294, 307 

of diphtheria antitoxin, 307 

of tetanus antitoxin, 294 
Urethritis, 258 
Urinary bladder, inflammation of, 340 
Urine, collection of, 104 


Vaccination, anthrax, 230, 285 
Asiatic cholera, 230, 366 
small-pox, 12, 230, 395 
typhoid, 230, 350 


SUBJECTS 487 
Vaccines, bacterial, 12, 230, 277 
Vaccinia, 12, 230, 395 
Vacuum, culture in, 129 
Vaginitis, gonorrheal, 260 
Van Ermengem’s flagella stain, 53 
Variola, 395 
Veillon culture method, 116, 129 
Vibrio, 5 
cholerz, 360 
Deneke’s, 367 
metchnikovi, 367 
of Finkler and Prior, 367 
tyrogenum, 367 
Vibrion septique (Cl. edematis), 286 
Vincent’s angina, 357 
spirillum, 357 
Vinegar, 177 
Violet, anilin-water gentian, 41 
gentian, 41 
methyl (pyoktanin), 78 
methylene, 44 
Virulence, 209 
factors influencing, 209 
loss of, in cultures, 119 
Virus, filterable, 13, 156, 388 
Visibility of microscopic objects, 25 
by light and shade, 26,* 27* 
by quality of light (color), 28 
Von Pirquet test, 323 
Vulvo-vaginitis, 260 


Warmth, 119 

Wasserman test, 382 

Water, bacteria, 185 
cholera germs in, 194, 366 
collection of samples, 103 
disinfection of, 189 
examination, 189 
filtration, 187 
intestinal bacteria in, 193 
self-purification of, 186 
storage of, 187 
typhoid bacilli in, 194 

Watery solution of aniline dyes, 40 

Weigert’s stain, 59 

Weil’s disease, 373 


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